Thailand Ministry of Public Health  

U.S. Centers for Disease Control and Prevention 

 

International Emerging Infections Program (IEIP) 

 

 

 

POPULATION-BASED SURVEILLANCE 

FOR MICROBIAL AGENTS OF PNEUMONIA AND SEPSIS 

 WITH DETECTION OF STREPTOCOCCUS PNEUMONIAE 

 

 

STANDARD OPERATING PROCEDURES 

FOR CLINICAL AND LABORATORY STAFF 

 

 

 

 

 

 

 

 



Document History 

 

First version prepared February 2005 (Version 1.0) 

Revised version prepared 02 September 2005 (Version 1.1) 

 

 

International Emerging Infections Program 

Thailand MOPH  U.S. CDC Collaboration 

Department of Diseases Control, 3rd Floor, Building 7 

Ministry of Public Health. Muang, Nonthaburi 11000 Thailand 

Tel: 02-5914039, 02-5912182 Fax: 02-5915753 

 

 

Document prepared by: 

Julie E. Fischer 

Sonja J. Olsen 

Wanna Wongjindanon 

Scott F. Dowell 

Leonard Peruski 

 

 

 


TABLE OF CONTENTS 

INTRODUCTION 4 
OVERVIEW OF HEMOCULTURE PROCESS 5 
QUALITY ASSURANCE AND QUALITY CONTROL 6 
A. CLINICAL METHODS 7 
1. Guidelines for obtaining blood cultures 7 
1.1 Clinical indications: identifying patients for blood culture 7 
2. Collection of Patient Specimens 9 
2.1 Precautions for safe collection of blood 9 
2.2 Blood culture supplies 10 
2.3 Good blood collection techniques 11 
2.4 Collecting blood for cultures 12 
3. Record-keeping and Reporting Results 15 
3.1 Record Keeping  Patient Information 15 
3.2 Record Keeping  Laboratory Information 16 
3.3 Record Keeping  Reporting Results 20 
4. Transport of clinical materials 24 
4.1 Post-collection process at district (community) hospitals 24 
4.2 Post-collection process at provincial (general) hospitals 26 
4.3 Transporting isolates and blood culture materials to IEIP laboratory 27 
5. Processing patient blood cultures and sera 28 
5.1 Preparing MB bottles for incubation 28 
5.2 Loading and removing bottles from BacT/ALERT 3D instrument 29 
5.3 Preparing patient serum disks for pre-hospital antibiotic use testing 31 
B. MICROBIOLOGICAL METHODS 33 
6.1 Preparing solid media and slides for initial identification of positive isolates 33 
6.2 Gram stain procedure for positive blood cultures (Hucker modification) 36 
6.3 Acid-fast bacilli staining for positive MB bottles 38 
6.4 Alarm-positive blood cultures with no apparent growth 40 
6.6 Confirmatory identification of S. pneumoniae 45 
6.7 Antimicrobial susceptibility testing of S. pneumoniae 48 
6.8 Confirmatory identification of H. influenzae 52 
6.9 Shipping clinically significant isolates for further testing 55 
APPENDIX I: QUALITY CONTROL AND QUALITY ASSESSMENT 57 
APPENDIX 2: TESTING PATIENT SERUM FOR PRE-HOSPITAL ANTIBIOTIC USE 66 
APPENDIX 3: PREPARING MEDIA AND REAGENTS 68 
References 81 
Introduction 


 

Twelve hospitals in the Thai province of Nakhon Phanom, on the Lao border, and 8 hospitals in the eastern Thai 
province of Sa Kaeo on the Cambodian border currently participate in a pneumonia disease surveillance network 
directed through a Thai Ministry of Public Health (MOPH) - U.S. Centers for Disease Control and Prevention (CDC) 
collaboration. This Thai-U.S. collaboration uses active surveillance to determine the population-based incidence of 
radiographically confirmed pneumonia. 

 

With the support of the PneumoADIP program at Johns Hopkins University and the Global Alliance for Vaccines 
and Immunization (GAVI), the Thai-U.S. CDC collaboration now aims to strengthen microbiology laboratory 
capacity in Nakhon Phanom and Sa Kaeo. This support will allow MOPH clinicians to request sensitive blood 
cultures as clinically indicated, with special emphasis on patients with suspected pneumonia and children with fever 
and possible bacteremia. This increased microbiology capacity will: 

 

 strengthen the Thai MOPH system for population-based pneumonia surveillance; 
 allow clinicians to meet the recommended standards of clinical care for pneumonia and sepsis; 
 help define the population-based incidence of invasive bacterial disease, including Streptococcus pneumoniae; in 
these two provinces; and 
 provide a profile of antimicrobial sensitivity for S. pneumoniae and other bacteria. 





Using pre-treatment blood cultures to identify disease etiology for patients who require hospitalization for community-
acquired pneumonia, as emphasized by internationally accepted practice guidelines, can improve care of individual 
patients by allowing selection of the most effective therapies. Microbiological diagnosis can also advance knowledge 
and improve care of other patients by detecting pathogens of potential epidemiological importance and patterns of 
antibiotic resistance, and can help physicians select antibiotics to limit development of microbial resistance in the 
community.1,2 

 

Streptococcus pneumoniae (or pneumococcus) affects children and adults worldwide, causing invasive diseases such as 
pneumonia, meningitis, and sepsis, and accounting for about two-thirds of all cases of community-acquired 
bacterermic pneumonia.3 An accurate assessment of the disease burden from pneumococcus in these two provinces 
will allow for better characterization of potential benefits from routine use of existing or new pneumococcal vaccines 
in Thailand. Therefore, the collection, transport, culture, and storage protocols described in this manual have been 
designed to collect potential pneumococcus isolates as efficiently as possible. The techniques adopted to preserve 
notoriously fastidious pneumococcus cultures for comprehensive analysis should allow for high-throughput, high-
sensitivity detection of a wide spectrum of invasive bacterial and mycotic diseases. 

 


 

BacT/ALERT Positive, no 
organisms stained or subcultured: 

Test with Binax NOW kit 

BacT/ALERT Positive as soon as alarm is detected:

Gram stain (all bottles) or AFB stain (MB bottles) positive cultures.

Subculture bacteria on blood, chocolate, and MacConkey agar. 

Identify organisms through standard microbiological testing. 

Complete antimicrobial sensitivity testing. 

Report positive hemoculture and staining results to ward nurse or 
physician and to district hospital laboratory immediately by phone. 

Send hard copy of results to district hospital laboratory via each 
hospitals surveillance officer. (Hospitals should expect final 
confirmatory results for general bacteria in =7 days, for fungi in =2 
weeks, and for mycobacteria in =2 months). 

Send isolates or culture bottles with yeasts, fungi, or unidentifiable 
organisms to IEIP to forward to Thai NIH. Send culture bottles 
with mycobacteria to IEIP to send to Thai TB Reference Center. 

Enter data from Specimen Collection Forms into microbiology 
database once each week. 

BacT/ALERT Negative: 

At 5 days, remove FA or PF bottles. 

At 42 days, remove MB bottle. 

Complete Specimen Collection Form. 

Notify laboratory by phone of negative 
aerobic bottles at 48 hours. 

Send hard copy of results at 5 days 
(FA/PF) and 42 days (MB) to district 
hospital laboratory for patients chart 
via each hospitals surveillance officer. 

In provincial laboratory on day of blood draw:

Check bottles for volume and color change. Subculture any yellow bottles immediately. 

Prepare MB bottles for incubation by adding 1 mL supplemental fluid. 

Scan bottle labels and enter patient information as prompted by BacT/ALERT 3D machine. 

Place hemoculture bottles in BacT/ALERT 3D machine. 

Soak filter disk with patient serum and freeze for later evaluation of pre-hospital antibiotic use. 

Update Specimen Collection Forms and place in laboratory logbook.

Transport samples in sealed, insulated containers from district to provincial laboratories: 

Maintain bottle temperature between 15-30C in district hospital laboratory. 

Record in laboratory logbook and transport at pre-arranged times daily via dedicated driver system. 

Store all bottles in provincial lab at 15-30C in designated site until bottles can be processed. 

Text Box: Overview of Hemoculture Process
Collection of blood for hemocultures (in district and provincial hospitals):

Observe precautions for safe collection of blood. 

Reduce potential contamination by cleaning venipuncture site thoroughly with 70% alcohol and 2% 
tincture of iodine (see blood draw poster). 

Collect blood at optimal volume for each bottle (2 bottles per patient) plus one serum tube. 

Record patient information and place bottle number stickers on Specimen Collection Form. 

Initiation of hemocultures (in district and provincial hospitals):

Ordered by MOPH physicians as clinically indicated. 

Blood drawn by MOPH clinical staff at admission for inpatient care (before antibiotic treatment). 

No consent required. 

All blood collection materials and Specimen Collection Form provided by IEIP.

Identify patients with clinical indications for hemocultures:

Adults and children of all ages with suspicion of pneumonia 

Children =5 years of age with fever and possible sepsis or bacteremia 



Quality Assurance and Quality Control 

 

Throughout these standard operating procedures (SOPs), boxed sections refer to Quality Control and Quality 
Assurance steps. These boxes serve as reminders of specific actions that must be taken to assure clinicians and 
patients that the laboratory results can be trusted. 

 

Quality Assurance (QA) can be defined as the overall system for assuring a reliable standard of work. QA includes 
internal quality control (defined below), comprehensive record-keeping to demonstrate the validity and performance 
of tests, training of personnel, monitoring of results to improve laboratory efficiency and reliability, and external 
assessments to test laboratory proficiency. QA exists not only to detect errors when they occur, but to improve the 
laboratory system continuously. A comprehensive QA program spans the entire laboratory process, from collection 
of specimens to feedback from clinical staffs. 

 

Quality Control (QC) refers to the steps that laboratory technicians take with every procedure to demonstrate that 
their tests provide reliable results. QC includes selecting the right assays, media, and reference control strains for every 
situation; assessing whether media, reagents, equipment, and assays perform correctly; including appropriate controls 
to confirm that microbiological techniques yield results that make sense and fall within normal parameters; maintaining 
essential environmental conditions in media quality, temperature, humidity, CO2 levels, and sterility; and keeping 
accurate records that allow technicians to share and validate their results quickly. 

 

Many comprehensive references to QA/QC systems exist. The American Society for Microbiology and the Clinical 
Laboratory Standards Institute (formerly NCCLS) offer numerous publications on general and specific QA/QC 
procedures. The Manual for the Laboratory Identification and Antimicrobial Susceptibility Testing of Bacterial Pathogens of Public 
Health Importance in the Developing World (World Health Organization, Geneva, 2003) describes QC practices for many 
common bacterial causes of pneumonia and sepsis. The World Health Organizations Southeast Asian Regional 
Office has also posted Blood Safety and Clinical Technology: Quality Assurance in Bacteriology and Immunology online at: 
http://w3.whosea.org/EN/Section10/Section17/Section53/Section375.htm. 


A. Clinical Methods 

 

1. Guidelines for obtaining blood cultures 





With the PneumoADIP/GAVI funding, IEIP will support the routine performance of blood cultures as clinically 
indicated in all 20 participating hospitals in Nakhon Phanom and Sa Kaeo. Clinicians are urged particularly to obtain 
blood cultures for all patients admitted for inpatient care with suspected pneumonia, and for all children five years of 
age and younger admitted with fever and possible sepsis or bacteremia. 

 

All blood cultures will be initiated at the request of hospital clinicians with materials, equipment, technical training, and 
staff support provided by IEIP. Neither the patient nor the hospital will incur any cost. Because Thai and 
international standards of care support blood cultures for diagnosis of community-acquired pneumonia and sepsis, 
and because the Thai MOPH performs pneumonia surveillance as a core function, written informed consent is NOT 
required to perform these blood cultures. A patient who receives a blood culture at the order of a hospital clinician 
with the support of IEIP resources may enroll subsequently in a research study requiring consent. 

 

 

1.1 Clinical indications: identifying patients for blood culture 

 

1) Blood cultures are indicated for patients of all ages who are admitted for inpatient care with suspected 
pneumonia. Suspicion of pneumonia, as per the case definition used by the IEIP pneumonia surveillance network, 
can be based upon one or more signs of acute infection and one or more signs and symptoms of respiratory illness, 
as described below. 

 

a) Signs of acute infection: 
o Fever (>38.2C within 24 hours, including by patient history) 
o Hypothermia (<35.5C within 24 hours, including by patient history) 
o Abnormal WBC 
o Abnormal differential 





b) Signs or symptoms of respiratory illness: 
o Cough 
o Sputum production 
o Hemoptysis 
o Chest pain 
o Dyspnea 
o Tachypnea 
o Abnormal breath sounds 







Because blood for cultures should be drawn prior to antibiotic treatment, clinicians should not wait for evidence of a 
new infiltrate on a chest radiograph to begin blood cultures for patients whose clinical signs and symptoms suggest 
pneumonia. 


2) Cultures should also be obtained for all children = 5 years of age admitted for inpatient care with fever 
of >38.2C or hypothermia (<35.5C) and suspicion of sepsis or bacteremia, as determined by the clinician. 
Clinical signs or symptoms of possible sepsis or bacteremia are often non-specific, especially in small infants, but may 
include one or more of the following:4 

 

o Altered mental state, including lethargy or irritability 
o Feeding difficulties 
o Vomiting 
o Convulsions 
o Signs of respiratory distress, including apnea or tachypnea 
o Pallor, cyanosis, or jaundice 


 





2. Collection of Patient Specimens 

 

2.1 Precautions for safe collection of blood5 

 

Infections may be transmitted between patients and staff during the blood-drawing procedure. Viral agents are the 
greatest hazard, and can be lethal. Of particular importance are the viruses causing hepatitis and acquired 
immunodeficiency syndrome (AIDS). The greatest risk to health-care personnel may come from samples from 
asymptomatic, and therefore unsuspected, carriers of the hepatitis B and human immunodeficiency viruses. To 
decrease the risk of transmission of these viral agents, the following recommendations should be followed as closely 
as possible: 

 

1. Wear latex or vinyl gloves impermeable to liquids, and change gloves for each patient. 



2. Use a new syringe and needle for each patient. 



3. Wipe the gloves with 70% isopropyl alcohol before drawing blood. 



4. Disinfect the rubber stopper of the blood culture bottles with 70% alcohol before and after puncturing the 
top with the needle. 



5. Inoculate the drawn blood into the culture bottle within one minute to prevent the blood from clotting in 
the syringe. Do not change needles before inoculating the blood culture bottles. 



6. Dispose of syringes and needles in a puncture-resistant, autoclavable container. No attempt should be made 
to re-cap any needle. 



7. Transport inoculated blood culture bottles in securely sealed, individual plastic bags. Specimen containers 
should be individually and conspicuously labeled. 



8. Remove gloves and discard in an autoclavable container. 



9. Wash hands with soap and water immediately after removing gloves. 



10. In the event of a needle stick, or other skin puncture or wound, wash the wound liberally with soap and 
water. Encourage bleeding. 



11. Immediately report any contamination of the hands or body with blood, or any puncture wound or cut to 
the supervisor and follow hospital guidelines for assessment and treatment. 



2.2 Blood culture supplies 

 

All blood culturing will be carried out using the automated BacT/ALERT 3D blood culture system and pre-
packaged non-vented blood culture media manufactured by BioMrieux. For each patient, two separate 
hemoculture bottles will be inoculated and incubated: 

 

 one aerobic medium (FAN for adults and children >5 or Pediatric FAN for children =5 years of age), to 
capture the majority of bacteria likely to cause invasive disease; and 



 one medium (BacT/ALERT MB) designed to capture slow-growing mycobacteria, as well as other bacteria, 
yeasts, and fungi. 





Most materials necessary to collect blood for cultures will be pre-packaged by IEIP staff in a sealable plastic bag to 
make the process as convenient as possible. Each bag will contain a form and tubes pre-labeled with the same 
running number. IEIP will supply gloves and cotton balls with sterilizing jars in bulk to each hospital. The IEIP 
microbiology laboratory technologists in the laboratories at Nakhon Phanom Hospital in Nakhon Phanom province 
and Crown Prince General Hospital in Sa Kaeo province will ensure that each district hospital receives an adequate 
supply of blood draw kits. These kits, which contain BacT/ALERT 3D blood culture media, should be stored at 
15-30C, away from direct sunlight. If an air-conditioned room is not available, the kits should be stored in an 
insulated cold chain box in an interior room (away from open hallways and windows). 

 

The main IEIP laboratory in Nonthaburi will supply two types of pre-packaged blood draw kits, one for children 
=5 years and one for adults and children >5 years. 

 

 

 

 

Blood collection should be carried out in the location and by personnel assigned by normal hospital procedures. 
The person drawing blood should gather everything needed for the process (blood culture supply kit, tourniquet, 
autoclavable puncture-proof container, gloves, and sterile cotton balls) before beginning. 

Text Box: Blood culture supply kit
Adults and children >5 years
Specimen Collection Form
1 sterile 20 cc syringe
1 sterile 22 gauge needle
1 sterile 23 gauge needle 
3 70% isopropyl alcohol packaged swabs 
1 2% tincture of iodine swab stick
Adhesive bandage
1 numbered red-top serum tube
1 numbered cryotube
1 MB blood culture bottle (black lid) 
1 FA blood culture bottle (pale green lid)
Text Box: Blood culture supply kit
Children ?5 years
Specimen Collection Form
1 sterile 10 cc syringe
1 sterile 23-gauge wing (butterfly) set
1 sterile 24-gauge wing (butterfly) set 
3 70% isopropyl alcohol packaged swabs
1 2% tincture of iodine swab stick
Adhesive bandage
1 numbered red-top serum tube
1 numbered cryotube
1 MB blood culture bottle (black lid)
1 PF blood culture bottle (yellow lid)

2.3 Good blood collection techniques 

 

The quality of blood collection techniques affects both the sensitivity and the reliability of any blood culture 
system.6,7 Success in detecting clinically significant pathogens relies upon:. 

 

The timing of the culture  blood should be drawn prior to administration of antibiotics. 

 

The volume of blood collected  inadequate blood volumes decrease the likelihood of detecting clinically 
significant infections in any blood culture system.8,9 Despite common assumptions that bacterial concentrations are 
higher in the blood of small children than adults, and reluctance to draw blood from younger patients, low-level 
bacteremias that might be missed by relying upon small blood volumes can be quite common in infants.10 Including 
two or more bottles per patient (rather than a single bottle) appears to increase yield regardless of whether the blood 
is collected from one or more sites, especially in children.11

 

Thorough skin disinfection at an appropriate blood draw site  contamination of blood cultures with bacteria 
that commonly reside on the skin can confuse patient care and consume resources. Thorough disinfection of the 
venipuncture site with a combination of alcohol and iodine solutions can significantly reduce the number of 
contaminated blood cultures, but only if used correctly.12,13 Skin should be disinfected first by scrubbing vigorously 
enough to create friction with a 70% isopropyl alcohol solution (higher concentrations are not effective and should 
not be used). An iodine solution should then be used and left on the skin for an appropriate length of time before 
and during the blood draw. A tincture of 2% iodine must be allowed to act for 30 full seconds prior to drawing 
blood, and iodophors such as 10% povidone-iodine require a full two minutes. Blood should always be drawn 
directly from a vein rather than through a central venous access catheter.14

 

The ratio of blood to culture medium in the blood culture bottle  the BacT/ALERT hemoculture system 
depends upon an optimal broth-to-blood ratio. The bottles contain not only concentrated nutrient broth, additional 
growth factors, and antibiotic-binding activated charcoal, but the anti-coagulant sodium polyanetholesulfonate 
(SPS). Inoculating the bottles with too little blood might not only exclude the possibility of detecting low-level 
bacteremias, but might inhibit bacterial growth due to insufficiently diluted media. Filling bottles beyond their 
optimal amount can lead to detection of CO2 emitted by blood cells rather than growing bacteria.15 Thus, under-
filling culture bottles can cause significant numbers of false negatives, while over-filling can lead to false positives. 

 


2.4 Collecting blood for cultures 





Blood cannot be transported before being placed in the BioMrieux culture bottles because the collection procedure 
does not use an anticoagulant. Once drawn, blood must be inoculated into the bottles within one minute to 
prevent clotting in the syringe. 

 

a) For adults and children > 5 years: collect a total of 15.5 mL: 
 10 mL to inoculate one BacT/ALERT FAN Aerobic (FA) bottle; 
 5 mL to inoculate one BacT/ALERT Mycobacteria Blood (MB) bottle; and 
 0.5 mL to fill one serum (red-top) tube for testing pre-hospital antibiotic use. 





b) For children = 5 years: collect a total of 7.5 mL: 
 4 mL to inoculate one BacT/ALERT Pediatric FAN (PF) bottle; 
 3 mL to inoculate one BacT/ALERT Mycobacteria Blood (MB) bottle; and 
 0.5 mL to fill one serum (red-top) tube for testing pre-hospital antibiotic use. 







Generally, it should be possible to draw this amount of blood, even from small children. IF it is impossible to 
draw enough blood to inoculate both bottles, use the volume drawn to fill the FA bottle to 10 mL (adults) 
or PF bottle to 4 mL (children = 5 years). Do not divide blood evenly among the two bottles. For example: 

 

Blood draw from 3 year-old = 5 mLs . 4 mLs in PF bottle + 0.5 mLs in serum tube 

 NOT 2.5 mLs in PF bottle, 2.5 mLs in MB bottle 

Blood specimens from a single draw can be divided for multiple uses if necessary, in which case the blood 
culture bottles should be inoculated before filling any other blood collection tubes. (NCCLS recommends 
filling tubes from a single blood draw in the following order: blood culture bottles, then citrate, serum, heparin, and 
finally EDTA tubes.16) 

 

 

 

The following procedure should be followed in collecting blood for hemocultures:17 

 

1. Explain the procedure to the patient or guardian. 



2. Complete the top (boxed) part of the Specimen Collection Form. 



3. Check that the hemoculture bottles are clear and the color indicator intact and blue-green. Label the bottles 
with the patients name, hospital number, and date. Do not write over the barcode or lot number. Remove 
and place the peel-off number from the FA or PF hemoculture bottle under Bottle Number on the front 
of the Specimen Collection Form, and the label from the MB bottle under Bottle Number on the back of 
the form. 



4. Check that the running number on the red-top serum tube (for example, N000001) matches the running 
number on the Specimen Collection Form. 



5. If any dirt is visible on the patients arm, clean with soap and water. 





___AcroPDFMTS

6. Select an arm and apply a tourniquet to restrict the flow of venous blood. The most prominent vein of the 
forearm is usually chosen. 



7. Disinfect the skin at the proposed venipuncture (needle insertion) site as follows: 



a. Scrub the venipuncture site vigorously with a 70% alcohol wipe for 30 seconds. 



b. Apply iodine solution from the packaged swab to an area approximately 5 cm in diameter around 
the venipuncture site for 30 seconds. 



c. Allow to dry completely. 




 

d. If the vein is palpated again, repeat the skin disinfection. 



e. For patients with iodine sensitivity, scrub with 70% alcohol alone for 60 seconds. 





8. Remove the protective flip-top cover from the hemoculture bottles. 



9. Carefully disinfect the top (rubber septum) of each hemoculture bottle with a 70% alcohol wipe and allow 
to dry for 1 minute before inoculation. 



10. Wipe the gloves with 70% alcohol to disinfect. 



11. Insert the needle bevel-upwards into the vein. Once the vein is entered, withdraw the required amount of 
blood by pulling back the plunger of the syringe at a slow, steady pace. Air must not be pumped into a vein. 
After the desired amount of blood is obtained, release the tourniquet and place a sterile cotton ball over the 
insertion site while holding the needle in place. Withdraw the needle and ask the patient or guardian to hold 
the cotton ball firmly in place until the wound has stopped bleeding. 



12. Immediately inject blood into the hemoculture bottles. DO NOT change needles after drawing blood. 
Swirl the bottle gently several times to mix. 



13. Inject 0.5 mLs of blood into the red-top serum tube. 



14. Dispose of the needle and syringe in a puncture-resistant, autoclavable container. Do not re-cap the needle. 



15. Clean the top of each hemoculture bottle and the serum tube with a 70% alcohol wipe. 



16. Clean the patients venipuncture site with 70% isopropyl alcohol, removing iodine to prevent skin irritation. 
Bandage and ask the patient or guardian to continue to place pressure on the upright arm for a few minutes. 



17. Empty the plastic bag of all unused supplies EXCEPT the labeled cryotube. 



18. Place the inoculated culture bottles, the red-top tube, and the Specimen Collection Form back into the 
plastic bag and re-seal. 



19. Transport the sealed bag to the clearly labeled designated temperature-controlled storage site (either an air-
conditioned room between 15-30C or an insulated storage box supplied by IEIP) in the hospitals 
microbiology laboratory. Bottles must never be refrigerated. If room temperature exceeds 30C, place a 
cold pack inside an adhesive bag (shipping label bag) stuck to the inside of the cold chain box lid, 
and then place the samples in the insulated box as far from the cold pack as possible. 



 




 

 




Ice pack in adhesive plastic bag 

Blood culture bottles 


3. Record-keeping and Reporting Results 

 

3.1 Record Keeping  Patient Information 

 

IEIP will provide Specimen Collection Forms for Hemocultures with two sections, one to be filled in by the person 
drawing blood, and the rest to be completed in the laboratory. All underlined fields on the forms will be used for 
patient care purposes only. 

 

1) The person performing the blood draw will complete the following information in the top (boxed) section: 

 

Information on Specimen Collection Form 

Purpose 

Patients name 

Clinical management 

Patients hospital number 

Clinical management 

Patients admission number 

Clinical management 

Name of the person drawing blood 

Clinical management 

Patients ward or hospital room location 

Clinical management 

Name of the physician requesting the hemoculture 

Clinical management 

Physicians phone number 

Clinical management 
(notification of results) 

Name of the hospital where the patient is admitted 

Clinical management and 
disease surveillance 

Date and time of specimen collection 

Clinical management and 
disease surveillance 

Patient age (in months if less than 1 year) 

Disease surveillance 

Diagnosis (possible pneumonia, possible sepsis, or other) 

Disease surveillance 

History of patients antibiotic use, if known 

Disease surveillance 



 

2) The person performing the blood draw will remove the peel-off part of the barcode from each hemoculture 
bottle, and place it into the appropriate section (FAN/Pediatric FAN or MB) on the Specimen Collection 
Form. One adhesive label with the running number will already have been attached to the Specimen 
Collection Form. 


 




 

___AcroPDFMTS

3.2 Record Keeping  Laboratory Information 



1) Blood for hemocultures will be collected in both district (community) and provincial (general) hospitals. 
Any inoculated bottles will be transported to the laboratory of the hospital in which they were collected. In 
the district hospitals, the bottles will then be picked up once each day by a driver, who will deliver them to 
the provincial hospital microbiology laboratory. The microbiology laboratory technician from the provincial 
hospital will phone the district hospital laboratory with the preliminary results, and then send a hard copy of 
the results to put into the patients chart. 


Good record-keeping in each laboratory is essential to track the cultures and the results throughout the cycle 
from district hospital to provincial hospital and back. Each hospital laboratory will keep a bound logbook 
(supplied by IEIP) in which the laboratory technician records information about blood culture bottles 
inoculated in that hospital. The laboratory technician who receives the specimens (and, later, the results) 
should record the following information in the blood culture logbook: 

 

 






Information for Blood Culture Logbook 

Running number 

Date and time of specimen arrival in laboratory 

Patients name 

Patients hospital number 

Patients admission number 

Patients ward or hospital room location 

Name of the physician requesting the hemoculture 

record date received and: 

For positive culture results  

Gram stain/AFB stain results 

Identification of isolate 

For negative culture results  
make a checkmark in the 
appropriate column to indicate: 

No growth at 48 hours 

No growth at 5 days (FA or PF 
bottle) or 42 days (MB bottle) 



 

 

2) Inoculated blood culture bottles will be transported to the provincial hospital microbiology laboratory 
(either directly after the blood draw or after transport from the district hospital). The laboratory technician 
who receives the inoculated cultures bottles will add the following information to the Specimen Collection 
Form: 



a) Check the box indicating whether the aerobic bottle is FAN or Pediatric FAN 
b) The time that the bottles were received in the provincial microbiology laboratory 
c) The time that the bottles were placed into the BacT/ALERT 3D incubator 
d) The approximate inoculation volume of blood added to each bottle, as compared visually to pre-
measured standard bottles. 





3) After recording this information, the laboratory technician will place the original form in chronological 
order in a binder supplied by IEIP. 



4) The laboratory technician who removes the bottles from the BacT/Alert 3D incubator and who processes 
the samples will complete the following information on the original form: 



 


e) The date and time that each bottle became positive, or an indication of no alarm 
f) Description of any organisms by Gram stain (positive FAN and MB bottles) 
g) Description of any organisms by AFB stain (positive MB bottles only) 
h) Description of subculture growth results 
i) Optochin sensitivity (for suspected S. pneumoniae isolates only) 
j) Identification  the suspected genus and species of positive cultures 
k) Antimicrobial sensitivity testing results (recorded by attaching the laboratory report) 
l) Date and time that the technician notified the hospital/physician of results 
m) Date that the technician shipped isolates to IEIP for further testing and storage 
n) The results of the patient serum bioassay for pre-hospital antibiotic use (to be determined later). 





Specimen Collection Form for Hemocultures 

 


To be filled out by phlebotomist: 

Patient name: _________________________ HN: ____________________ AN: ______________________ 

 

Blood drawn by: _______________________ Physician name: __________________ Phone: _____________ 

 

Hospital name: ________________________________ Patient Ward/Room: ____________________________ 

 

Patient age: ____ years ____ months (if <1 year) Date/Time of blood draw: ___/____/____ ___:___ 

 

Diagnosis: . Possible pneumonia . Possible sepsis (= 5 years only) . Other______________ 

Has patient received any antibiotics within 72 hours of blood draw? . Yes . No . Dont know 

Name of antibiotic(s): 1) _____________________ 2) _____________________ 3) _________________________ 

Blood drawn using: . needle/wing set from blood culture kit . IV catheter .other (please specify)______________________ 

 

To be filled out by lab technician: 

Received in NP Microbiology Lab: Date ____/____/_____Time ___:___ by ________________ 

Placed in BacT/ALERT machine: Date ____/____/_____Time ___:___ by ________________ 

 

. Pediatric FAN (4 mL) or . FAN Aerobic (10 mL) -- one bottle per patient 

Bottle Number 

Blood volume 

Positive alarm (date and time) 

At 5 days 

 

 

 

 

mLs

 

_____ /_____ _____ : _____ 

 

. No alarm



 

Removed from BacT/ALERT machine: Date ____/____/_____Time ___:___ by __________________________ 

Culture and Gram stain results 

Gram stain 1 

 Gram stain 2 

Gram reaction 

. Positive 

. Negative 

. Not found 

 

 

 

 

 Shape 

. Bacilli 

. Cocci 

. Other_____ 

 

 

 

 

Arrangement 

. Pairs 

. Chains 

. Clusters 

. None 

. Other______ 

Gram reaction 

. Positive 

. Negative 

. Not found 

 

 

 

 

 Shape 

. Bacilli 

. Cocci 

. Other_____ 

 

 

 

 

Arrangement 

. Pairs 

. Chains 

. Clusters 

. None 

. Other______ 

Culture results 

. No growth 

. Growth on BAP 

. Growth on CAP 

. Growth on MAC 

 

Hemolysis 

. None 

. Alpha 

. Beta 

 

Optochin-sensitive 

. Yes 

. No 

. Not tested 

 

 

Culture results 

. No growth 

. Growth on BAP 

. Growth on CAP 

. Growth on MAC 

 

Hemolysis 

. None 

. Alpha 

. Beta 

 

Optochin-sensitive 

. Yes 

. No 

. Not tested 

 

 



 

Gram stain 3:______________________________________________________________________________________ 

 

Plated: Date ____/____/_____Time ___:___ Plates examined: Date ____/____/_____Time ___:___ 

 

Identification (Genus/Species): 1)__________________________2)___________________________3)________________________ 

 

Reported by: __________________________________ Approved by: ______________________________________________ 

Hospital/physician notified: Date ____/____/_____Time ___:___ 

Isolate sent to IEIP: Date ____/____/_____ 

PLEASE ATTACH ALL MICROBIOLOGY REPORTS WITH ANTIBIOTIC SUSCEPTIBILITY 

Text Box: Running number
Text Box: Please collect patients blood before antibiotic administration

Specimen Collection Form for Hemocultures (Page 2) 

 

HN: ____________________ Patient name:________________________ Hospital name: _____________________ 

 

Mycobacteria (MB) Bottle -- one bottle per patient 

(Children = 5 years = 3 mL, children and adults >5 years = 5 mL) 

 

Bottle Number 

Blood volume 

Positive alarm (date and time) 

At 42 days 

 

 

 

mLs

 

_____ /_____ _____ : _____ 

 

. No alarm



 

Removed from BacT/Alert machine: Date ____/____/_____Time ___:___ by ________________________________ 

 

Gram stain results 

Gram stain 1 

 Gram stain 2 

Gram reaction 

. Positive 

. Negative 

. Not found 

 

 

 

 

 Shape 

. Bacilli 

. Cocci 

. Other_____ 

 

 

 

 

Arrangement 

. Pairs 

. Chains 

. Clusters 

. None 

. Other______ 

Gram reaction 

. Positive 

. Negative 

. Not found 

 

 

 

 

 Shape 

. Bacilli 

. Cocci 

. Other_____ 

 

 

 

 

Arrangement 

. Pairs 

. Chains 

. Clusters 

. None 

. Other______ 

Culture results 

. No growth 

. Growth on BAP 

. Growth on CAP 

. Growth on MAC 

 

Hemolysis 

. None 

. Alpha 

. Beta 

 

Optochin-sensitive 

. Yes 

. No 

. Not tested 

 

 

Culture results 

. No growth 

. Growth on BAP 

. Growth on CAP 

. Growth on MAC 

 

Hemolysis 

. None 

. Alpha 

. Beta 

 

Optochin-sensitive 

. Yes 

. No 

. Not tested 

 

 



 

Gram stain 3:______________________________________________________________________________________ 

 

Acid-fast Bacilli (AFB) stain results 

. Negative 

. AF Bacilli positive 

. Other AFB forms (yeast/fungi) 

All AFB + samples should be sent to IEIP for subculture and identification 



 

Plated: Date ____/____/_____Time ___:___ Plates examined: Date ____/____/_____Time ___:___ 

 

Identification (Genus/Species): 1)______________________2)_______________________3)_____________________ 

 

PLEASE ATTACH ALL MICROBIOLOGY REPORTS WITH ANTIBIOTIC SUSCEPTIBILITY 

 

Reported by: ______________________________ Approved by: ________________________ 

Hospital/physician notified: Date ____/____/_____Time ___:___ 

Isolate sent to IEIP: Date ____/____/_____ 

 

Disk test for serum antimicrobials 

Zone of inhibition: 

. None 

. Positive __________ mm 



Binax test for S. pneumoniae Ag 

. Positive 

. Weakly positive 

. Negative 

. ND 



 

 

Text Box: Running number

3.3 Record Keeping  Reporting Results 





Prompt feedback to clinicians, whether results are positive or negative, is a critical factor in both 
good clinical practice and in establishing strong trust in the microbiology laboratorys reliability and 
utility. The following system outlines directions for communication between the provincial 
microbiology laboratory, the district hospital microbiology laboratories, and the clinicians who 
requested the hemocultures. The IEIP microbiologist in each province will be responsible for 
ensuring that clinicians receive preliminary and confirmatory results in a timely way. 

 

Reporting positive results: 

 

1) The results of the Gram staining should be recorded on the Specimen Collection Form. 



2) Once isolates from a positive blood culture have been identified by Gram stain, the 
provincial laboratory microbiologist should immediately report the preliminary results 
(positive blood culture, and Gram reaction and morphology of the isolate) by phone to the 
ward nurse or the physician who requested the blood culture, by clinician preference. 



3) At this time, the microbiologist should explain to the ward nurse or physician that more 
information confirming the identity of the organism will be available in 24 hours and the 
antimicrobial susceptibility testing results will be available in 48 hours. The microbiologist 
should explain that all results will be sent to the hospitals microbiology laboratory by phone 
call and then in a hard copy via the driver system (to place in the patients chart) within 
another day. The hard copy of the results of confirmatory testing for general bacteria will 
arrive within one week (or 2 weeks for fungi or yeast, or 2 months for mycobacteria). 



4) After notifying the clinician, the provincial laboratory microbiologist should report 
preliminary results by phone to the district microbiology laboratory. The laboratory 
technician in the district microbiology laboratory should note the preliminary results in the 
bound blood culture logbook. For patients in the provincial hospital, the results should be 
recorded in the provincial hospitals bound blood culture logbook. 



5) Once the results of overnight growth on solid media and standard microbiological testing 
have confirmed the identity of the isolate, the microbiologist should repeat steps 1-4 above. 
(Report the results by phone to the ward nurse or physician, then to the district 
microbiology laboratory by phone to be recorded in the bound logbook.) 



6) The original Specimen Collection Form should be completed, removed from the binder, 
copied 2 times, and returned to the same chronological order in the binder. 



a) For patients in the provincial hospitals, one copy should be sent directly to the ward 
(according to hospital procedure) to be placed in the patients chart. 



b) For patients in the district hospitals, one copy should be sent via the driver system to the 
hospitals surveillance officer, who will bring the copy to the hospital laboratory to be 
placed in the patients chart according to hospital procedure. 



c) The second copy should be placed in a folder to send to IEIP for later data entry. 








7) The results of antimicrobial sensitivity testing should be recorded on the standard form used 
regularly by the provincial hospitals microbiology laboratory. (In Nakhon Phanom and Sa 
Kaeo, this may be hand-written or a print-out from existing clinical microbiology software.) 



8) The antimicrobial testing results should also be copied twice. The original report should be 
attached to the original Specimen Collection Form in the provincial laboratorys binder. The 
second copy should be sent to the ward or to the district hospital microbiology laboratory to 
place in the patients chart, and the third copy should be attached to the copy of the patients 
Specimen Collection Form in the IEIP data folder. 



9) If the second bottle becomes positive, the process above should be repeated EXCEPT that 
it is not necessary to phone the clinician or ward nurse if the organism appears to be the 
same by Gram stain, growth on solid media, and other standard microbiological testing. 





 

Reporting negative results: 

 

1) The provincial laboratory microbiologist should report negative (no-growth) culture results 
by phone to the district microbiology laboratory after 48 hours of culture for PF and FA 
bottles. The laboratory technician in the district microbiology laboratory should note the 
results in the bound specimen log-book. 



2) The district microbiology laboratories (and the provincial microbiology laboratories for 
patients in the provincial hospitals) should notify clinicians of negative results through 
normal hospital procedures at 48 hours and 5 days. 



3) The original Specimen Collection Form indicating No growth should be completed, 
removed from the binder, copied twice, and returned to the same chronological order in the 
binder. 



a) For patients in the provincial hospitals, one copy should be sent directly to the ward 
(according to hospital procedure) to be placed in the patients chart. 



b) For patients in the district hospitals, one copy should be sent via the driver system to the 
hospitals surveillance officer, who will bring the copy to the hospital laboratory to be 
placed in the patients chart according to hospital procedure.. 



c) The extra copy should be placed in a folder to send to IEIP for later data entry. 


 






Capturing results for the Pneumonia Surveillance Network 

 

1) The IEIP Microbiology Database will contain no personally identifiable information. 



2) Every Friday, the laboratory microbiologist in each provincial laboratory will download the 
records captured by the BioMerieux BacT/ALERT 3D systems Observa software, and use 
the IEIP Microbiology Database Access forms to enter the remaining information from 
Specimen Collection Forms completed that week. IEIP staff in Nonthaburi will provide 



support for converting Observa files into entries that can be completed using Microsoft 
Access. 



3) Every Tuesday, the laboratory microbiologist should send the folder with copies of all 
completed Specimen Collection Forms and antimicrobial sensitivity reports to the IEIP 
office in Nonthaburi. The data will be entered a second time at IEIP. 



4) Each hospitals IEIP disease surveillance officer already identifies cases of suspected 
pneumonia by routinely screening inpatient charts. The disease surveillance officer will be 
able to determine whether a patient who meets the case definition for pneumonia had a 
blood culture by looking for a completed Specimen Collection Form in the patients chart. 
The Pneumonia Surveillance Network database contains fields for entering blood culture 
results as part of regular data entry. (The Specimen Collection Form may not have been 
placed in a patients chart by the time that the disease surveillance officer reviews it for the 
first time. Regular reviews of the bound blood culture logbooks in each hospitals laboratory 
combined with review of post-discharge patient charts should capture these results.) 



5) The accuracy and completeness of data captured for the Pneumonia Surveillance Network 
(which should include hemoculture results for every patient with clinical signs and symptoms 
of pneumonia) and the GAVI Microbiology Database (which should include the results of 
all hemocultures conducted for patients with suspected pneumonia or children =5 years of 
age with fever and possible sepsis, without any personally identifiable information) will be 
validated by regular audits using the original Specimen Collection Forms and patient 
logbooks as references. 



 


Overview of Record-Keeping and Reporting

At the time of blood draw:

 Phlebotomist completes patient and hospital information on 
Specimen Collection Form. 

 Phlebotomist places peel-off bottle number stickers from each 
blood culture bottle on Specimen Collection Form. 

 Phlebotomist places Specimen Collection Form in bag with 
samples to send to Microbiology Laboratory 

Text Box: Provincial hospitals
Negative culture bottles 

At 48 hours post-incubation: 

 Provincial laboratory microbiologist 
reports no growth results by phone to 
district hospital laboratories; results 
recorded in bound logbook by district 
laboratory technician. 

 Microbiologist reports no growth results 
for patients in provincial hospitals to wards 
per regular hospital procedures. 

At 5 days post-incubation: 

 Microbiologist reports no growth by 
phone to district hospital laboratories; 
results recorded in bound logbook by 
district laboratory technician. 

 Microbiologist sends copy of Specimen 
Collection Form marked no growth 
directly to ward or to district hospital 
laboratory by driver to be placed in 
patients chart. 

District hospitals 

Positive culture bottles 

After alarm and Gram stain of isolate: 

 Date/time and Gram stain results noted on 
Specimen Collection Form. 

 Provincial laboratory microbiologist 
reports preliminary Gram stain results by 
phone to ward nurse or physician. 

 Microbiologist reports preliminary results 
by phone to district hospital laboratories; 
results recorded in bound logbook by 
district laboratory technician. 

After standard microbiological testing: 

 Results and identity of organism noted on 
Specimen Collection Form. 

 Microbiologist reports results (identity of 
organism) to ward nurse or physician and 
district hospital laboratory by phone; 
results recorded by laboratory technician. 

 Microbiologist sends copy of Specimen 
Collection Form to ward or to district 
hospital laboratory by driver/surveillance 
officer for patients chart. 

After antimicrobial sensitivity testing: 

 Results copied, sent to ward or district 
hospital laboratory for patients chart, and 
attached to Specimen Collection Form. 

In provincial microbiology laboratory:

 Laboratory technician completes bottle 
arrival and volume information on Specimen 
Collection Form. 

 Specimen Collection Form placed in 
laboratory binder. 

In district microbiology laboratory:

 Enter patient information into bound 
logbook. 

 Send original Specimen Collection 
Form with specimens to provincial 
hospital microbiology laboratory.

Once per week:

 Data entered into Access database on-site. 

 One copy of completed form sent to IEIP 


4. Transport of clinical materials 

 

S. pneumoniae and some other common disease agents of pneumonia and sepsis, such as Haemophilus 
influenzae, are fastidious and fragile bacteria. The ability to culture and identify S. pneumoniae 
successfully can be complicated by the tendency of densely grown cultures to undergo autolysis or 
self-destruction. Delaying the entry of blood culture bottles into the BacT/ALERT 3D instrument 
for up to 12 hours post-inoculation falls within the manufacturers normal operating parameters and 
requires no special precautions, as long as the inoculated bottles can be maintained at 15-30C.18 
Therefore, the following process has been developed to protect potential S. pneumoniae isolates 
against extremes of temperature or incubation past the log phase of growth that might cause false 
negatives and lost cultures. 

 

 

4.1 Post-collection process at district (community) hospitals 

 

All blood culture bottles inoculated at the district (community) hospitals will be transported to the 
provincial hospital microbiology laboratory by car at pre-assigned times for incubation and 
identification of any isolates through standard microbiological techniques. 

 

The IEIP provincial Surveillance and Research Coordinator will oversee the transportation system. 
Drivers will pick up bottles daily at a scheduled time and place coordinated by the director of the 
district hospital microbiology laboratory, the hospitals IEIP surveillance officer, the Surveillance 
and Research Coordinator, and the driver. Together, this team will decide: 

 

 What time the driver will arrive each day, based on his route and the goal of collecting the 
most freshly drawn cultures possible; 
 Who in the district hospital will meet the driver each day to transfer the insulated box 
containing the sealed bags of inoculated hemoculture bottles and blood tubes to the car, and 
to carry fresh media, the empty insulated box, and hard copies of completed Specimen 
Collection Forms back to the microbiology laboratory; and 
 Who in the district hospital will be responsible for calling the driver and the provincial 
hospital laboratory if there are NO inoculated bottles for transport. 





The microbiology laboratory technician in each provincial hospital should contact the district 
hospital laboratories if expected shipments do not arrive. The IEIP microbiology laboratory 
technician should also arrange for the driver to pick up the empty cold chain transport boxes, new 
hemoculture media bottles, and copies of completed Specimen Collection Forms to transport to the 
district hospitals. 

 

Inoculated hemoculture bottles should be protected from temperature extremes (<15C or >30C) 
prior to and during transport in an insulated cold chain box, supplied by IEIP. The inoculated 
hemoculture bottles must never come into contact with a cold pack, or be placed in a refrigerator. 
IEIP will supply thermometers to measure ambient room temperatures. The district laboratory 
technician should note and record the room temperature daily at the warmest time of the day. If the 
insulated box will sit where temperatures exceed 30C, place a cold pack in the adhesive plastic 
bag inside the lid of the cold chain box. All inoculated blood culture bottles should be received 
by the provincial microbiology laboratory between 14:00 and 15:00 each day. 


 

 

 

Checklist for drivers

 

_____ Each driver has an assigned weekday route clearly mapped by the IEIP Surveillance 
and Research Coordinator, with expected distance in kilometers indicated. 

 

_____ Each driver has a plan to: 

 

 Begin at the provincial microbiology laboratory every Monday  Friday between 
11:00 and 12:00 (depending on the length of the route) to pick up supplies and 
completed Specimen Collection Forms for each district hospital laboratory; 
 Travel to the most distant hospital on the route first to drop off supplies and 
pick up patient specimens, and then stop by each subsequent hospital on the way 
back to the provincial hospital; 
 Drop off supplies and pick up patient specimens at each district hospital 
laboratory; 
 Keep supplies and patient specimens between 15-30C in the passenger 
compartment of the vehicle with air-conditioning on, stopping for the minimum 
necessary amount of time at each district hospital; 
 Deliver the patient specimens, in their insulated cold boxes, to the provincial 
hospital microbiology laboratory by 14:00 (Sa Kaeo) or 15:00 (Nakhon Phanom) 
each day. 





_____ Each driver has a mobile phone where he/she can be contacted by the district 
hospital laboratories. The district hospital laboratory technician must notify the 
driver if there are no patient specimens on a weekday (Monday  Friday). . 

 

_____ A monthly schedule assigns one driver in each province to be on call for picking 
up patient specimens by request each Saturday and Sunday. Each laboratory has a 
copy of the current drivers schedule and contact information. 

 

_____ The driver in each province scheduled to be on call on weekends has a mobile 
phone where he/she can be contacted by the district hospital laboratories. On 
Saturday and Sunday, the district hospital technician must request a patient specimen 
pick-up by calling the driver before the normal pick-up time. 

___AcroPDFMTS

4.2 Post-collection process at provincial (general) hospitals 

 

Drivers will deliver all inoculated blood culture bottles from the district hospitals to the provincial 
hospital laboratories each day. Clinicians at the two general hospitals will also be encouraged to 
request blood cultures for patients seen on-site, which may represent as many as one-third of all 
blood culture specimens collected in each province. 

 

The chief of each provincial hospitals microbiology laboratory should designate a specimen 
receiving site for inoculated cultures in an air-conditioned room (15-30C). IEIP will supply 
thermometers, to be calibrated yearly, in order to measure ambient room temperatures. If room 
temperatures exceed 30C, the specimens should be stored in an insulated cold chain box. 
Inoculated culture bottles should never come into direct contact with a cold pack or be placed in a 
refrigerator. 

 

The Specimen Collection Form should be kept with the inoculated bottles until the laboratory 
technician records the date and time received, date and time placed in the BacT/ALERT 3D, the 
approximate blood volume, and the type of aerobic bottle (FAN or Pediatric FAN), and then the 
form should be placed in the three-ring binder. 

 

All inoculated bottles should be placed in the BacT/ALERT 3D instrument just before the close of 
normal laboratory working hours. For every patient specimen, a disk should be prepared using the 
serum in the red-top tube and the disk should be frozen for later testing (see section B.6.1 below for 
details). 

 

 


4.3 Transporting isolates and blood culture materials to IEIP laboratory 





Once the blood culture process and standard microbiological testing have been completed in the 
provincial hospital microbiology laboratory, three types of samples will need to be shipped to the 
IEIP laboratory in Nonthaburi for preservation or further testing: 

 

 Positive blood cultures (by alarm) that cannot be identified conclusively through 
standard microbiological testing in the laboratory. The provincial laboratories should 
ship the original blood culture bottle to IEIP, along with Gram stains and the isolate 
inoculated into two nutrient agar stabs and onto two Dorset Egg media slants if it has been 
successfully cultured on solid media (see Microbiology section 6.8 for details). Specimens 
should be accompanied by a copy of the original Specimen Collection Form and the 
Specimen Referral Form for the Thai National Institutes of Health (NIH). IEIP will 
forward these to the Thai NIH for identification and antibiotic susceptibility testing. 


If no organisms can be identified by Gram stain or isolated on solid media from a blood 
culture bottle despite a positive alarm, the bottle should be tested using the modified Binax 
NOW Streptococcus pneumoniae Test protocol (see Microbiology Section 6.5). 

 


 Any clinically significant isolates from positive blood cultures that have been 
identified (see Section 6.8 for an explanation of clinical significant pathogens for shipping). 
The provincial laboratories should ship any Gram stains, as well as the isolate inoculated into 
two nutrient agar stabs and onto two Dorset Egg media slants. These should be labeled with 
the running number, patient name, and hospital ID number, listed on the Specimen 
Referral Form/Bacterial isolate list for confirmatory identification, and sent in batches to 
IEIP every Tuesday along with a shipment cover sheet. 


IEIP will forward these to the Thai NIH for preservation at -70C in TSB media containing 
15% glycerol and further testing at the discretion of the Thai MOPH (all S. pneumoniae 
isolates will be serotyped at CDC-Atlanta and tested for penicillin susceptibility using the E-
test, while a selection of other specimens will be tested for confirmatory identification as a 
QC method). 

 


 Positive blood cultures that contain acid-fast bacilli, yeasts, or fungi, as determined 
by initial staining and microscopy. The provincial laboratories should ship the original 
blood culture bottle and any Gram- or AFB-stained organisms fixed on slides, but should 
not attempt to culture such organisms on solid media. Specimens should be shipped with a 
copy of the Specimen Referral Form for the Thai National TB Reference Laboratory Center 
or Thai NIH (as appropriate) attached. IEIP will forward suspected mycobacteria to the 
Thai National TB Reference Laboratory Center and yeasts or fungi to the Thai NIH for 
confirmatory identification and antibiotic sensitivity testing. 





Specimens should be packed according to international safety guidelines. All specimens should be 
clearly labeled. Bottles and tubes should be placed in a sealed plastic secondary container, packed 
into a specimen box to protect the specimens during transport; and finally placed in an external 
cardboard box shipping box. IEIP will supply all shipping containers, labels, and forms. 

 


5. Processing patient blood cultures and sera 

 

Once patient specimens have been received in the provincial microbiology laboratories, from 
patients at either the same site or from the district hospitals, the following steps must be taken: 

 

1. Recording of specimen and patient information in the bound blood culture logbook and/or 
on Specimen Collection Forms (see Sections 3 and 4 above). 
2. Preparing MB bottles for incubation by supplementing with MB/BacT Enrichment Fluid. 
3. Loading blood culture bottles into the BacT/ALERT 3D automated blood culture machine. 
4. Preparing a patient serum disk to use in evaluating the impact of pre-hospital antibiotic use 
on the sensitivity of the blood culture system. 





Several of these steps can be completed at the same time with careful attention to keeping the 
Specimen Collection Forms with the blood culture bottles until all information has been recorded. 
For example, the red-top serum tubes for the last step could be placed in the centrifuge to spin while 
the laboratory technician completes the Specimen Collection Forms and the laboratory logbook. 
The MB bottles can be prepared for incubation in the biosafety cabinet at the same time assembly 
line-style, with the technician changing needles carefully between bottles. 

 

 

5.1 Preparing MB bottles for incubation 

 

To ensure optimal growth of mycobacteria, all inoculated MB bottles must be supplemented with 1 
mL of MB/BacT Enrichment Fluid, supplied by BioMrieux in vials with a total fluid volume of 5.5 
mLs each, prior to incubation. Inoculated MB bottles can sit at room temperature (15-30C) for up 
to 24 hours prior to the addition of MB Enrichment Fluid, although delayed supplementation can 
lead to concomitant delay in time to detection of positive organisms.19 

 

To supplement inoculated MB bottles: 

 

1. Place inoculated MB bottles in a biosafety cabinet to avoid accidental aerosolization of 
potentially infectious materials. Use standard precautions for safe handling of blood. 



2. Wipe gloves with 70% isopropyl alcohol, then carefully disinfect the top (rubber septum) of 
each MB bottle and each MB/BacT Enrichment Fluid vial with 70% isopropyl alcohol. 



3. Using a sterile needle and 1 cc syringe, aseptically transfer 1 mL of MB/BacT Enrichment 
Fluid into one MB bottle. 



4. Discard the needle and syringe to prevent cross-contamination of hemoculture bottles. 



5. Repeat this process until all inoculated MB bottles have been supplemented and then load 
the inoculated, supplemented MB bottles into the BacT/ALERT 3D instrument. 


 





5.2 Loading and removing bottles from BacT/ALERT 3D instrument 

 

The operators manual to the BacT/ALERT 3D automated blood culturing machine, provided by 
BioMrieux, contains full instructions for operating the instruments hardware and software. The 
following information highlights only an overview of operating procedures and QC issues. 

 

The BacT/ALERT 3D system detects bacterial growth through continuous monitoring of a 
chemical reaction in which the rising levels of CO2 emitted by multiplying microorganisms causes a 
fall in pH, and the subsequent change of the colorimetric indicator from blue-green to light green or 
yellow. The instrument uses the same algorithm to detect growth in both aerobic and mycobacterial 
media for the first four days (allowing detection of rapidly growing bacteria in the MB bottles), then 
switches to a more sensitive mechanism for detecting the growth of slow-growing organisms. Once 
the instrument has been calibrated to 35C and set to the correct parameters, daily use should 
involve the following steps (within the operating guidelines provided by BioMrieux). 

 

 

 

Loading bottles: 

 

1. Press the Load Bottles button on the main screen. 



2. Use the barcode reader to scan the first bottle. The instrument will determine the type of 
bottle, and whether it falls into the 5-day (FA or PF) or 42-day (MB) protocol. 



3. Enter the hospital ID number, accession number, running number, hospital name, and 
patients first and last name in the fields on the screen. 



4. Open a drawer and insert the bottle, bottom-first, into any empty cell (indicated by a light). 



5. Go to the next bottle. Repeat until all bottles are loaded. 



6. Check that all incubator drawers are closed and press the Check button. 


 




Removing bottles: 

 

1. The instrument will indicate, by audible alarm and a change in screen color, when a bottle 
has become positive. Generate an Unload Report from the main screen and locate the 
Specimen Collection Forms that go with each bottle to be unloaded. 



2. From the main screen, press the Unload Positives button. Green lights will indicate the 
drawers that contain positives, and lights will also indicate the positive cells within each 


___AcroPDFMTS

drawer. Remove each positive bottle and scan with the barcode reader again. Note the time 
and date of positive alarm on the Specimen Collection Form, and proceed with subculture 
and staining. Positive bottles should be removed as promptly after the indication of a 
positive alarm as possible, and processed immediately. 



3. Finish unloading the positive bottles and press the Check button. 



4. From the main screen, select the Unload Negatives button. Lights will indicate the 
appropriate drawers and cells, and those bottles can be removed and scanned with the 
barcode reader. For negative bottles, check No alarm at 5 days for FA or PF media, or 
No alarm at 42 days for MB media, on the correct Specimen Collection Form. Negative 
bottles should still be treated as potentially infectious waste, and autoclaved prior to disposal. 



5. IF the BacT/ALERT 3D instrument becomes completely full, remove the oldest MB bottles from 
the instrument and continue incubating in a standard 35C incubator for the remainder of 
the 42 days. Any bottles removed in this fashion will need to be Gram- and AFB-stained 
and subcultured, whether or not color change occurs. 





 

___AcroPDFMTS

5.3 Preparing patient serum disks for pre-hospital antibiotic use testing 

 

The patients use of antibiotics (either self-prompted or at the direction of private clinicians) prior to 
hospital admission can have a significant impact on the sensitivity of a blood culture system.20 The 
prevalence of pre-hospital antibiotic use in the Nakhon Phanom and Sa Kaeo in patients presenting 
with possible pneumonia or sepsis is unknown. As a QC measure to evaluate whether pre-hospital 
antibiotic use will regularly affect the yield and sensitivity of blood cultures in these two provinces, 
serum collected at the time of blood culture inoculation from all patients will be used to test for the 
presence of antibiotics. 

 

IEIP will supply the 6-mm paper filter disks in autoclave-ready sleeves of 20 disks each, marked with 
autoclave indicator tape. The sleeves should be autoclaved by the provincial microbiology 
laboratories as needed, and carefully re-sealed after aseptic removal of disks with sterilized forceps. 

 

1. Remove the red-top serum tube and cryovial from the bag containing the inoculated blood 
culture bottles. Confirm that running number on the red-top serum tube matches the 
running number on the cryovial and the Specimen Collection Form. 



2. If the clot has contracted sufficiently to remove 20 L of serum easily with a pipet tip, 
continue. If not, centrifuge the red-top serum tube(s), with appropriate balance tubes, for 10 
minutes with no braking in a swinging bucket centrifuge at 1300 x g or less. 



3. Line up all red-top serum tubes and corresponding cryovials in the biosafety cabinet to avoid 
accidental aerosolization of potentially infectious materials. Use standard precautions for safe 
handling of blood. 



4. Remove one 6-mm paper filter disk per patient aseptically from the autoclaved sleeve of 
disks using sterilized forceps, and place on a sterile surface in front of each tube and vial set. 



5. Remove the stopper from the first patients red-top serum tube. Aspirate 20 L of serum 
from the separated blood and dispense onto the paper filter disk. Repeat for each patient. 



6. Air-dry all disks for 5 minutes. 



7. Using sterile blunt-edged forceps, place each dried serum-soaked filter disk into the correct 
cryovial and secure the top. 



8. Store all patient serum disks at -70C for later testing of pre-hospital antibiotic activity as a 
QC measure (see Appendix 2). 



 

 


 

 

 

___AcroPDFMTS
___AcroPDFMTS
___AcroPDFMTS
___AcroPDFMTS
___AcroPDFMTS
___AcroPDFMTS
___AcroPDFMTS
Text Box: Overview of culture process in provincial laboratories

B. Microbiological Methods 

 

In order to determine the burden of invasive bacterial and mycotic disease through routine blood 
cultures, laboratory staff in the provincial hospital laboratories must be able to isolate bacteria from 
positive culture bottles on solid media, and subculture isolates for confirmatory identification and 
antimicrobial sensitivity testing.21 

 

The procedures that follow begin by outlining techniques common to all potential isolates, but focus 
primarily on identifying the importat causes of invasive bacterial disease S. pneumoniae, H. influenzae 
and N. meningitidis. This section does not comprehensively address techniques to identify other 
bacteria that may be of clinical importance. Microbiologists should refer to clinical microbiology 
manuals such as the American Society for Microbiologys Manual of Clinical Microbiology (Washington, 
DC, USA; 1992) or the World Health Organizations Basic Laboratory Procedures in Clinical Bacteriology 
(Geneva; 2001) for other identification procedures. 

 

The appearance and growth characteristics of isolated bacterial colonies, combined with the 
microscopic experience of a Gram-stained specimen, often allow preliminary identification of an 
isolate. Gram stains of colonies and broths may be especially helpful if atypical or aberrant reactions 
are observed on the tests to identify H. influenzae and S. pneumoniae. S. pneumoniae are lancelate gram-
positive diplococci, sometimes occurring in short chains, and H. influenzae are small gram-negative 
rods with random arrangement. A more comprehensive microbiology manual should be consulted 
for Gram stain reactions of other bacteria. 

 

 

6.1 Preparing solid media and slides for initial identification of positive isolates 

 

After a positive bottle has been signaled and unloaded from the BacT/ALERT 3D machine, and the 
Specimen Collection Form completed appropriately, a small amount of media from the blood 
culture bottle will be aspirated to inoculate solid media, perform a Gram stain (for FAN, Pediatric 
FAN, and MB bottles) and perform an acid-fast bacillus (AFB) stain for positive MB bottles only. 

 

All media plates for isolating samples on solid media will be purchased from Clinag (Clinical 
Diagnostics, Ltd., Bangkok, Thailand), and supplied with appropriate documentation of QC testing 
for each batch. Plates should be stored at 4C until ready for use. All plates should be examined for 
signs of visible contamination prior to use; IEIP technicians should be notified of any media plates 
that demonstrate contamination prior to opening, or fail internal QC testing. 

 

1. For every positive blood culture bottle, prepare the following by labeling each with the 
patients name, hospital number, and the date: 



a. One clean (unused) glass slide for Gram stain; 
b. A second clean (unused) glass slide for AFB stain (MB bottles only); 
c. One sheep blood agar plate (pre-warmed to room temperature); 
d. One chocolate blood agar plate (pre-warmed to room temperature); and 
e. One MacConkey agar plate (pre-warmed to room temperature). 





2. Place positive blood culture bottles in a biosafety cabinet to avoid accidental aerosolization 
of potentially infectious materials. Use standard precautions for safe handling of blood. 



3. Wipe gloves with 70% isopropyl alcohol, then carefully disinfect the top (rubber septum) of 
each bottle with 70% isopropyl alcohol. 



4. Using a sterile needle and syringe, aseptically remove 0.5 mLs of blood culture media from 
one positive bottle. 



5. Inoculate 100 L of blood culture onto each of the three solid media plates and streak to 
isolation using a flamed sterile inoculating loop. 



6. Place one drop of blood culture media onto each clean glass slide as indicated and allow to 
air-dry in the biosafety cabinet. 



7. Invert and incubate media plates for up to 48 hours (or until the growth of colonies is 
observed) at 35C, placing the blood and chocolate agar plates in a 5% CO2 incubator. 



8. Store the original blood culture bottle at room temperature until bacterial growth has been 
confirmed by Gram stain and subculture. Bottles should be kept for at least one week. If 
no organisms can be subcultured from the blood culture bottle despite a positive alarm, 
follow the instructions in Appendix 3 for using the Binax NOW S. pneumoniae antigens kit. 






 

 

 

___AcroPDFMTS

6.2 Gram stain procedure for positive blood cultures (Hucker modification) 

 

Positive cultures from either the aerobic (FAN or Pediatric FAN) or the mycobacterial (MB) bottle 
for each patient should be Gram stained as part of preliminary identification of isolates. Gram 
staining divides bacteria into two groups based on the composition of their cell walls: those that 
retain the primary stain crystal violet and appear purple on examination (Gram positive) and those 
that are de-colorized by an ethanol rinse and instead take up the counterstain, appearing pink on 
examination (Gram negative). Gram staining also increases the visibility of bacterial morphology, 
making it easier to identify the shape of an isolate. 

 

Gram staining should always be performed using a clean new slide. Debris from previous tests may 
remain on washed and re-used slides, making identification of organisms difficult, and can falsely 
suggest multiple isolates in a single bottle. Overheating during the flaming process, excessive 
washing with acetone, or attempting to stain old cultures can also confuse results, as any of these 
may result in Gram positive bacteria staining poorly and appearing to be Gram negative. 

 

All Gram stain reagents will be provided by IEIP. If commercially prepared reagent kits include 
specific instructions, follow these. Otherwise, take the following steps: 

 

1) Pass the slide with the air-dried drop of blood culture medium quickly through a flame 
three times to fix the smear. Flame only briefly. Slide should not be hot enough to cause 
discomfort when touched to back of hand. Alternatively, bacteria can be fixed using 
methanol (95%-100%). 



2) Flood the smear with ammonium oxalate-crystal violet and let stand for 1 minute. 



3) Rinse gently with tap water by running a gentle stream across the edge or back of the slide. 
Drain off excess water by tapping the slide on a paper tower. 



4) Flood the smear with Grams iodine solution and let stand for 1 minute. 



5) Rinse gently with tap water and drain. 



6) Decolorize with 95% ethyl alcohol for 5-10 seconds. 



7) Counterstain with safranin for 20-30 seconds, or with carbol-fuchsin for 10-15 seconds. 



8) Rinse the slide with tap water and blot dry. 



9) Examine the stained smear microscopically, using a bright-field condenser and an oil-
immersion lens. NOTE: The BioMrieux blood culture media contain activated charcoal 
particles, intended to bind antibiotic factors in blood that might impair bacterial growth. 
These may be aspirated from the culture bottle with the isolates and be deposited on the 
slide. These particles will appear dark, and may be mistaken for heavily stained Gram 
positive organisms initially. A negative control slide should be prepared by Gram stain 
techniques using un-inoculated BioMrieux blood culture media and compared to slides 
from positive blood cultures to help identify the charcoal particles more easily. 






10) Describe the observed organisms by morphology (shape) and color (indicating Gram 
positive or negative). The majority of organisms will fall into a few categories: 





Color 

Shape 

Arrangement 

Purple (Gram positive) 

Cocci (spheres) 

Bacilli (rods) 

Coccobacilli (intermediate 
forms) 

 

Chains 

Pairs 

Clusters 

None 

Pink (Gram negative) 



 

11) Record observations on Specimen Collection Form and use to guide confirmatory 
identification steps. If observed organisms do not fall into the categories above, select 
Other and describe the appearance. 



12) Slides should be kept until colony growth and the microscopic appearance of organisms 
from an isolated colony grown on solid media can be described and confirmed (at least one 
week). 





 

 

___AcroPDFMTS

6.3 Acid-fast bacilli staining for positive MB bottles 





For all MB bottles that signal a positive alarm in the BacT/ALERT 3D machine, the laboratory 
technician should prepare two slides: one for Gram staining, and the other for AFB staining to 
detect mycobacteria. MB bottles that become positive within 72 hours of inoculation most likely 
contain typical bacterial pathogens, but bottles that become positive after a much longer period (up 
to 42 days post-inoculation) should be suspected of containing slow-growing mycobacteria (M. 
tuberculosis, M. avium, or other mycobacterial pathogens), yeast, or fungi. These isolates should be 
treated as potentially highly infectious. 

 

IEIP will supply reagents for AFB staining through the Ziehl Neelsen technique. If commercially 
prepared reagent kits include specific instructions, follow these. Otherwise, take the following 
steps:22 

 

1) Pass the slide with the air-dried drop of blood culture medium through a flame three times 
to fix the smear. Flame for 3-4 seconds each time. 



2) Place the slide, smear side up, on a staining rack over a heat source. 



3) Flood the smear with filtered 1% carbol fuchsin. 



4) Heat the slide from below until vapors start to rise. Do not let carbon fuchsin boil, or slide 
dry completely. Continue to heat for up to five minutes. 



5) Allow the slide to cool completely (5-7 minutes). 



6) Rinse the slide gently with tap water to remove excess carbol fuchsin stain and drain (the 
blood culture smear will still appear red). 



7) Decolorize by flooding the slide with 25% sulphuric acid. Let the sulphuric acid sit on the 
slide for 2-4 minutes. 



8) Rinse gently with tap water and drain. 



9) If the slide still appears red, repeat the decolorization process, leaving sulphuric acid on the 
slide for 1-3 minutes and rinsing gently with tap water. 



10) Counterstain with 0.1% methylene blue solution for one minute. 



11) Rinse gently with tap water and drain. 



12) Examine the stained smear microscopically, using a bright-field condenser and an oil-
immersion lens. NOTE: The BioMrieux blood culture media contain activated charcoal 
particles, intended to bind antibiotic factors in blood that might impair bacterial growth. 
These may be aspirated from the culture bottle with the isolates and be deposited on the 
slide. A negative control slide should be prepared by AFB stain techniques using un-
inoculated BioMrieux blood culture media and compared to slides from positive blood 
cultures to help identify the charcoal particles more easily. 






13) Describe the observed organisms by morphology (shape) and color (indicating AFB positive 
or negative). The majority of organisms will fall into a few categories: 





Color 

Shape 

Arrangement 

Pink or red (acid-fast) 

Bacilli (rods) 

Cocci (spheres) 

Coccobacilli (intermediate 
forms) 

Branched or hyphae 
(suggesting yeast or fungi) 

 

Chains 

Pairs 

Clusters 

None 

Blue (negative) 



 

14) Record observations on Specimen Collection Form, determining whether organisms can be 
classified as acid-fast bacilli, other acid-fast forms, or negative. 



15) All AFB negative samples that appear to be bacteria by morphology and Gram stain should 
be processed as per standard bacterial protocols. All acid-fast bacilli and suspected yeasts or 
fungi should be shipped to IEIP in the original culture bottles. These will be forwarded to 
the Thai National TB Reference Center (mycobacteria) or the Thai NIH (fungi or yeasts), 
for further testing. These should not be sub-cultured in the provincial laboratories. 
Clinicians should be notified that positive specimens appear to be acid-fast bacilli 
(mycobacteria), yeast, or fungi, and that confirmatory identification and testing will be 
performed by one of the two reference laboratories. 





 

 

 

___AcroPDFMTS

6.4 Alarm-positive blood cultures with no apparent growth 





Occasionally, the BacT/ALERT 3D machine will signal that an inoculated blood culture bottle has 
become positive, but no organism can be identified by Gram stain or isolated on solid media. If 
positive standard reference strains included for QC purposes show that all reagents and media are 
performing as expected, and that conditions are optimal for bacterial growth (see QC references in 
sections 6.1, 6.2, and Appendix 1), three main possibilities should be considered: 

 

1. A false positive has occurred due to over-filling of the bottle, temperature fluctuations 
affecting the media or color indicator, or machine error; 



2. The culture has yielded an extremely fastidious and hard-to-identify organism that might be 
viable in small numbers (more likely if the alarm happens late in the incubation period); 



3. An isolate of S. pneumoniae has been cultured past the log phase of growth, resulting in a real 
alarm but inability to locate or culture organisms due to autolysis (self-destruction) of the 
previously viable isolate. 





To test whether an apparent false positive culture represents an autolysed S. pneumoniae isolate, IEIP 
will supply the NOW Streptococcus pneumoniae Test manufactured by Binax (Portland, Maine, USA). 
This rapid immunochromatographic assay is designed to detect S. pneumoniae antigens in the urine of 
patients with pneumonia and cerebrospinal fluid from patients with meningitis, but the test can be 
modified to detect pneumococcal antigens in blood culture medium even in no organisms can be 
Gram stained or cultured.23 Binax NOW kits should be stored between 15-30C. 

 

This test should be used for every apparent false positive blood culture bottle. If the results are 
positive, the laboratory microbiologist should report a preliminary identification of S. pneumoniae to 
the clinician who ordered the blood culture and the district hospital laboratory, and explain that no 
further testing will be possible. This result should also be recorded on the Specimen Collection 
Form, and sent to the patients chart through the normal mechanisms. 

 

 

Modified Binax NOW S. pneumoniae Test protocol for apparent false positive blood cultures: 

 

1) Place blood culture bottles in a biosafety cabinet to avoid accidental aerosolization of 
potentially infectious materials. Use standard precautions for safe handling of blood. 



2) Allow blood cultures to equilibrate to room temperature (15-30C) and swirl gently to 
resuspend antigens before beginning tests. 



3) Wipe gloves with 70% isopropyl alcohol, then carefully disinfect the top (rubber septum) of 
each bottle with 70% isopropyl alcohol. 



4) Unwrap one testing device for each specimen to be tested and lay flat in biosafety cabinet 
without touching the reaction area of the testing device. 



5) Remove one Binax swab per sample from the kit, and use the foil package from the testing 
device as a tray for the swab. Do not use other swabs for this test. 



6) Using a sterile needle and syringe, aseptically remove 0.5 mLs of blood culture media from 
each positive bottle. 



7) Drop culture media from the needle onto a Binax swab until the swab head is completely 
soaked, but not lying in a puddle of excess media. If the swab head drips when picked up, 
remove the excess liquid by pressing against inside edge of the foil package. 



8) Insert the swab into the bottom hole (swab well) on the inner right panel of the testing 
device. Firmly push upwards so that the swab tip is fully visible in the top hole. Do not 
remove the swab. 



9) Hold the Reagent A vial vertically (straight up and down) 1-2 cm above the device. Slowly 
let 3 drops of Reagent A fall into the bottom hole. 



10) Immediately remove the adhesive liner from the right edge of the test device, and close and 
seal the device. Repeat all steps for each apparent false positive bottle. 



11) Read the result in the window 15 minutes after closing the device. Results read after 15 
minutes may not be accurate; strongly positive samples may produce a visible sample line in 
less than 15 minutes. 



12) One or two lines should appear in the window on the testing device. A single pink-to-purple 
colored Control Line in the top half of the window means that the test was performed 
correctly, but no pneumococcus antigens were detected. The appearance of two pink-to-
purple colored lines, the Control Line and a Sample Line, indicated a positive result even if 
the sample line is very faint. If no lines appear, or only the bottom Sample Line appears, the 
test results are not reliable. If this happens, the test should be repeated using three samples: 
the pre-packaged positive and negative control swabs, and the blood culture again. 


To use the positive and negative control swabs with the Binax NOW Tests: 

 

13) Unwrap one testing device for each specimen to be tested and lay flat in biosafety cabinet 
without touching the reaction area of the testing device. Unwrap the prepackaged negative 
control swab and insert it into the bottom hole of a testing device. Firmly push upwards 
until the swab tip is fully visible in the top hole. Do not remove the swab. Repeat with a 
positive control swab and another testing device. 



14) Hold the Reagent A vial vertically (straight up and down) 1-2 cm above the device. Slowly 
let 6 drops of Reagent A fall into the bottom hole. 



15) Immediately close and seal the device. 





Negative 
result 

Positive 
result 

Invalid 
result 

Invalid 
result 

Control line 

Sample line 


16) Read the result in the window 15 minutes after closing the device. Results read after 15 
minutes may not be accurate; strongly positive samples may produce a visible sample line in 
less than 15 minutes. 





 

___AcroPDFMTS

6.5 Presumptive identification of S. pneumoniae, H. influenzae, and N. meningtidis 
isolates24 





The primary purpose of this section is to aid in preliminary identification of isolates of S. pneumoniae, 
H. influenzae, and N. meningitidis from positive blood cultures. The methods described here will not 
apply to other suspected bacterial agents of pneumonia and sepsis; clinical microbiology textbooks 
and manuals referenced in the introduction to this section provide detailed protocols for isolating 
and characterizing the range of other possible pathogens. 

 

Presumptive identification of these three organisms can be made on the basis of differential growth 
on blood and chocolate agar, macroscopic examination of colonies, and microscopic examination of 
Gram-stained organisms. After growth is observed on solid media inoculated from the positive 
blood culture bottles, a second Gram stain should be prepared from each pure culture through the 
following steps: 

 

1) Place one drop of physiological saline or distilled water on a clean, unused slide labeled with 
the patients name, hospital ID number, and the date. 



2) With a flamed sterile inoculating loop, touch the center of an isolated bacterial colony from 
one of the three solid media plates. 



3) Add the bacteria from the inoculating loop to the saline or distilled water with a gentle tap, 
using the loop to mix the organisms into suspension. 



4) Spread the suspension and allow to air dry for approximately ten minutes. 



5) Follow the steps for Gram staining described in section 6.3 (above). 





Presumptive identification of S. pneumoniae 

 

S. pneumoniae appear as Gram positive pairs of cocci (diplococci) or chains of cocci on microscopic 
examination. When examined macroscopically on solid media, S. pneumoniae appears as small, 
greyish, moist (sometimes mucoid), watery colonies with a greenish zone of a-hemolysis 
surrounding them on blood and chocolate agar plates. Some serotypes appear more mucoid than 
others, an effect exaggerated by fresh culture medium. Young pneumococcal colonies appear raised, 
and can be difficult to differentiate from a-hemolytic viridans streptococci, which also produce a 
greenish zone of hemolysis on a blood or chocolate agar plate. As the culture ages 24-48 hours, the 
colony becomes flattened and the central part of each colony becomes depressed, which does not 
occur with the viridans streptococci. A hand lens or microscope (30X-50X) can prove useful in 
distinguishing pneumococci from viridans streptococci. 

 

Presumptive identification of H. influenzae 

 

H. influenzae appear as small, Gram-negative bacilli or coccobacilli on microscopic examination. 
When examined macroscopically on chocolate agar plates, H. influenzae appears as large, flat, 
colorless-to-grey opaque colonies on chocolate agar. No hemolysis or discoloration of the medium 
is apparent. Encapsulated strains appear more mucoidal than non-encapsulated strains, which 
appear as compact grayish colonies. 


Presumptive identification of N. meningitidis 

 

N. meningitidis appear as Gram-negative, coffee bean-shaped diplococci on microscopic examination. 
When examined macroscopically on blood agar plates, young colonies of N. meningitidis are round, 
smooth, moist, glistening and convex, with a clearly defined edge. Some colonies appear to coalesce 
with other nearby colonies. Growth of N. meningitidis on blood agar appears greyish and 
unpigmented; older cultures become more opaquely grey and sometimes cause the underlying agar 
to turn dark. Well-separated colonies can grow from about 1 mm in diameter in 18 hours to as large 
as 4 mm, with a somewhat undulating edge, after several days. 

 

N. meningitidis grows on blood agar, whereas H. influenzae will not grow without supplements found 
in chocolate agar. When grown on chocolate agar, H. influenzae and N. meningitidis look similar. 
However, H. influenzae gives off a pungent smell of indol, while N. meningitidis does not. 

 

 


Growth on 

Gram stain 
morphology 

Presumptive 
identification

Confirmatory tests 

Chocolate 
agar 

Sheep blood 
agar 

+ 

+ 

Gram  

coffee bean-shaped 
diplococci 

N. meningitides 

Kovacs oxidase test, 
carbohydrate 
utilization test 

+ (a-hemolysis) 

+ (a-hemolysis) 

Gram + 

diplocci or cocci 

S. pneumoniae 

Optochin sensitivity, 
bile solubility 

+ 

- 

Gram  

small pleomorphic 
bacilli/coccobacilli 

H. influenzae 

X and V factor 
dependency, serum 
agglutination 



 

The Thai NIH will confirm identification of all potential isolates of N. meningitides, S. pneumoniae, and 
H. influenzae (see Section 6.9).

Positive blood culture

Inoculate solid media 

Perform Gram stain

Growth on: 

 Blood agar? 

 Chocolate agar? 

 MacConkey agar? 

Colony appearance? 

Positive or negative?

Shape/arrangement:

 bacilli? 

 cocci? 

 coccobacilli? 

a, , or no hemolysis? 

Chains/clusters/pairs?


6.6 Confirmatory identification of S. pneumoniae 

 

Confirmatory identification of pneumococcus follows the presumptive identification, described 
above, based on the microscopic identification of Gram-positive cocci in pairs or chains, and the 
macroscopic appearance of the small, grayish, and mucoid colonies demonstrating a-hemolysis on 
blood and chocolate agar plates incubated in a 5% CO2 atmosphere. 

 

Laboratory differentiation between S. pneumoniae and viridans streptococci can be accomplished by 
optochin and bile solubility testing. Pneumococci are susceptible to optochin and are bile soluble, 
while viridans streptococci are not. The bile solubility test can be particularly helpful when the 
results of the optochin susceptibility test appear ambiguous. 

 

Performance of the optochin susceptibility test 

 

1) Touch the suspect a -hemolytic colony with a sterile bacteriological loop and streak onto a 
blood agar plate in a straight line. Because the inoculum is streaked in a straight line, several 
3-4 colonies can be tested on the same plate, streaked in parallel lines and properly labeled. 



2) Aseptically place an optochin or P disk with a diameter of 6 mm (and containing 5 g 
ethylhydrocupreine) on the end of the streak where the wire loop was first placed. 



3) Incubate the plates in a CO2 incubator or candle-jar at 35C for 18-24 hours. 



4) Read, interpret, and record the results on the Specimen Collection Form: 



a) a-hemolytic strains with a zone of inhibition of growth greater than 14 mm in diameter 
(or >16 mm in diameter if using a 10-mm optochin disk) are considered susceptible to 
optochin, and are therefore pneumococci; 



b) a-hemolytic strains with no zones of inhibition are considered resistant to optochin, and 
are viridans streptococci; 



c) a-hemolytic strains with zones of inhibition ranging from 9 to 13 mm show intermediate 
resistance to optochin, and should therefore be tested for bile solubility. 


 

 







Oval: P 


Oval: P
Oval: P
Text Box: Diagram of optochin susceptibility test showing three streaks (three different strains) with varying susceptibilities, as described above.. 
Text Box: a
b
c
Text Box: b
b
c
Text Box: c
b
c

Performance of the bile solubility test

 

The bile solubility test is performed on isolates with small zones of inhibition in the optochin 
sensitivity test. It can be performed using either the tube method or the plate method. 

 

1) Using the tube method, two tubes are required for bile solubility testing of each suspect 
strain of S. pneumoniae. 



2) Take a loop of the suspect strain from fresh growth on a blood agar plate and prepare a 
bacterial cell suspension in 0.5 ml of sterile saline. The suspension of cells should be equal 
to that of a 0.5 McFarland density standard (see Appendix 3). 


 

a) If growth on the optochin test plate is sufficient, the suspension can be made with 
the bacterial cells collected from the specific streak of suspect S. pneumoniae. 

 

b) When there is insufficient growth to make a suspension of the proper density in 0.5 
mL of sterile saline, inoculate a blood agar plate with the suspect growth and 
incubate overnight at 35C in a 5% CO2 incubator to prepare a fresh culture. 

 

3) Divide the suspension into two equal amounts (0.25 ml per tube). Add 0.25 ml of saline to 
one tube and 0.25 ml of 2% deoxycholate (bile salts) to the other. 



4) Shake the tubes gently and incubate them at 35 - 37C for up to 2 hours 


. 

5) Examine the tubes periodically for lysis of cells in the tube containing the bile salts. A 
clearing of the tube, or a loss in turbidity, is a positive result. 



6) For the plate method, place a drop of 10% sodium deoxycholate solution directly on a 
colony of a freshly prepared culture of the suspect isolate. (To prepare the 10% solution of 
bile salts, add 1 g of sodium desoxycholate bile salts to 10 mL of sterile saline.) 



7) Keep the plate at room temperature (18-25C) or place it agar-side up on a level surface in an 
ambient air (non-CO2) incubator at 35C for approximately 15 minutes, or until the 10% 
bile salt reagent dries. 



8) When the reagent is dry, read, interpret, and record the results. 





Pneumococcal colonies are bile soluble and will disappear or appear as flattened colonies, while bile-
resistant streptococcal colonies will be unaffected. 

 

 

___AcroPDFMTS

Optochin susceptibility 

(diameter of zone of inhibition 
with 6-mm, 5 g optochin disk) 

Bile solubility 

Most likely identification 

Susceptible (=14 mm) 

Not necessary to test 

S. pneunomiae 

Intermediate/definite (9-13 mm) 

Yes 

S. pneunomiae 

Intermediate/small (=8 mm) 

No 

Viridans streptococci 

Resistant (no zone of inhibition) 

Not necessary to test 

Viridans streptococci 



 

 

Serotyping S. pneumoniae isolates 

 

The Quellung reaction is traditionally used to identify the serotypes of pneumococcal isolates due to 
its ease of use, speed, and accuracy. A Quellung reaction results when a type-specific antibody 
bound to the pneumococcal capsular polysaccharide causes a change in the refractive index of the 
capsule so that it appears swollen and more visible. The pneumococcal cell stains dark blue and is 
surrounded by a sharply demarcated halo, which represents the outer edge of the capsule. The light 
transmitted through the capsule appears brighter than either the pneumococcal cell or the 
background. Single cells, pairs, chains, and even clumps of cells may have Quellung reactions. 
However, most laboratories do not type pneumococcal isolates because of the large number of 
diagnostic antisera required. 

 

All confirmed isolates of S. pneumoniae identified through the routine use of blood cultures for 
patients with suspected pneumoniae, or in children ages 5 and under with fever and possible sepsis, 
in Nakhon Phanom and Sa Kaeo will be identified through the Quellung typing technique. These 
tests will be performed by technicians from the Thai NIH using facilities and reagents at the CDC 
(Atlanta, Georgia, U.S.) as per existing arrangement. S. pneumoniae isolates will be shipped to the 
IEIP laboratory each Tuesday (described in Sections 4.3 and 6.8), and transported to the Thai NIH 
for cryopreservation. Twice each year, these samples will be shipped to CDC and will be serotyped 
there using the Quellung reaction by Thai MOPH researchers who have already received training 
and conduct these tests routinely with IEIP support. 

 


6.7 Antimicrobial susceptibility testing of S. pneumoniae 

 

The results of antimicrobial susceptibility testing can be used to help make recommendations for 
clinical treatment for individual patients, and to help predict which antibiotics might be effective for 
patient populations in general. The disk diffusion method presented here represents a modification 
of the Kirby-Bauer technique standardized by NCCLS, as well as the Etest (AB BIODISK, Solna, 
Sweden) antimicrobial gradient agar diffusion technique for testing sensitivity to penicillin. If 
performed precisely according to the following protocol, this method will yield data that can reliably 
predict the in vivo effectiveness of specific antimicrobial agents against S. pneumoniae. 

 

The WHO recognizes three classes of antimicrobial susceptibility: susceptible, resistant, and 
intermediate. These can be used to describe the results of the disk diffusion testing. All results 
from antimicrobial susceptibility testing should be attached to the original Specimen Collection 
Form for each patient, and included when entering results each week into the GAVI database using 
Microsoft Access. The results should also be reported to the ward nurse or physician and the 
district hospital laboratory immediately by phone as soon as they are interpreted. 

 

IEIP will provide reagents necessary to perform the disk diffusion method  valid for certain 
antibiotics  and the Etest to gather data about the minimal inhibitory concentration (MIC) of 
penicillin for bacterial isolates. 

 

 

 

___AcroPDFMTS

Antimicrobial sensitivity testing by disk diffusion 

 

Mueller-Hinton agar medium supplemented with 5% sheep blood, poured at a uniform depth of 3-4 
mm, is the recommended medium for determining the antimicrobial sensitivity of S. pneumoniae 
isolates by disk diffusion. 

 

Susceptibility testing with penicillin disks does not provide reproducible results. Results of screening 
with the 1 g oxacillin disk can be generalized across the -lactam drugs for S. pneumoniae, allowing 
the technician to conclude whether a strain is susceptible (but not resistant) to penicillin. If the 
oxacillin disk produces a zone of inhibition of less than 20 mm, additional MIC testing by Etest 
must be performed to determine whether the isolate is resistant or susceptible to penicillin. 

 

1) Suspend viable colonies from a fresh pure culture of S. pneumoniae (grown overnight on 
blood or chocolate agar) in Mueller-Hinton broth or sterile physiological saline. 



2) Adjust the cell suspension to the density of a 0.5 McFarland turbidity standard (described in 
Appendix 2), carefully avoiding froth or bubbles while mixing. Compare the density of the 
suspension to the 0.5 McFarland turbidity standard by holding the suspension and the 
McFarland standard in front of a light against a white background with black lines. Add 
more saline or broth if the density appears too heavy, and more bacteria if the density 
appears too light. 



3) Dip a sterile cotton swab into the bacteria suspension, removing excess fluid by rotating 
against the sides of the dilution tube. 



4) Use the same swab (without re-dipping in the bacterial cell suspension) to inoculate the 
entire surface of the 5% sheep blood-Mueller Hinton agar three times, rotating the plate 60 
degrees between each inoculation to achieve even coverage. 



5) Allow the inoculum to dry before placing disks on the plates (5-15 minutes). 



6) Place the antimicrobial disks on the plates with a dispenser or sterile forceps, tapping them 
gently to ensure that they adhere to the agar. Once a disk contacts the agar surface, 
diffusion of the drug begins immediately, and the disk should not be moved. For suspected 
S. pneumoniae isolates, four disks can be placed on each plate. 



7) Incubate the plates in an inverted position (upside down) at 35C in a 5% CO2 incubator for 
20-24 hours. 



8) Measure the diameter of the zone of inhibition with a rule or calipers, measuring the distance 
from the last visible colony on each side of the disk from the top surface of the plate with 
the lid removed. Care should be taken not to touch the disk or surface of the agar with the 
ruler or calipers. 



9) Interpret the antimicrobial susceptibility of the isolate according the NCCLS performance 
standards, checking to be sure that the results for the positive control reference strain S. 
pneumoniae ATCC 49619 lie within the acceptable control range. 


 





 

 


 

Etest for MIC testing of S. pneumoniae 

 

Disk diffusion testing with oxacillin and S. pneumoniae isolates allows the laboratory to screen for 
susceptibility to penicillin, but does not allow technicians to distinguish whether strains that appear 
not to be susceptible exhibit complete or intermediate resistance. Therefore, all S. pneumoniae should 
also be tested for penicillin susceptibility using the commercially available Etest to determine MIC. 
All reagents will be supplied by IEIP, and the tests will be performed at the Thai NIH reference 
laboratory. 

 

Etests to determine penicillin resistance in S. pneumoniae isolates should be performed using 
Mueller-Hinton agar supplemented with 5% sheep blood. 

 

1) Suspend viable colonies from a freshly grown overnight blood agar plate in a Mueller-
Hinton broth tube, adjusting the suspension to the equivalent of a 0.5 MacFarland 
turbidity standard. 



2) Dip a sterile cotton swab into the bacterial suspension, removing excess fluid against the 
side of the tube, and inoculate the entire surface of the 5% sheep blood Mueller-Hinton 
agar plate evenly with the same swab, rotating the plate 60 degrees after each inoculation 
to ensure confluent growth. 



3) Allow the plate to dry completely (5-15 minutes). While the plate is drying, remove the 
Etest strips from the -20C freezer and allow the strips that will be used to warm to 
room temperature. Unused strips must always be stored at -20C. 





Subculture identified isolates 
on blood or chocolate agar 

Suspend bacterial inoculum in sterile 
saline or Mueller-Hinton broth 

Adjust suspension to equivalent of a 0.5 
MacFarland standard 

Inoculate a 5% sheep blood Mueller-
Hinton agar plate evenly with swab. 

Place disks on plate with sterile forceps.

Measure zones of inhibition with ruler or calipers.


4) Place the Etest strip onto the inoculated agar plate with sterile forceps. Make sure that 
the printed MIC values face upward. Once applied, do not move the antimicrobial strip. 



5) Incubate the plates in an inverted position (upside down) at 35C in a 5% CO2 incubator 
for 20-24 hours, following the manufacturers instructions. 



6) MICs are read by examining the intersection of the ellipse-formed zone of inhibition 
with the value printed on the Etest strip, read using oblique light, with a magnifying 
glass if necessary. The intersection should be determined from the point of inhibition of 
all growth, including hazes and isolated colonies. Follow the manufacturers instructions 
in the package insert for interpreting and reporting antimicrobial susceptibility results. If 
the intersection of the printed MIC value and the zone of inhibition lies between two 
markings, read the next highest standard value. 



7) Interpret the antimicrobial susceptibility of the isolate, checking to be sure that the 
results for the positive control reference strain S. pneumoniae ATCC 49619 lie within the 
acceptable control range. 





 

 

 

___AcroPDFMTS

6.8 Confirmatory identification of H. influenzae 

 

Confirmatory testing for H. influenzae follows the identification of small, pleomophic Gram-negative 
bacilli or coccobacilli which grow on chocolate but not blood agar plates and have a pungent indol 
smell. Laboratory testing for H. influenzae includes testing for X and V factor requirements and 
serotyping using an agglutination reaction. In the absence of vaccination, most invasive H. influenzae 
disease is caused by organisms with the type b polysaccharide capsule (H. influenzae b, or Hib). 

 

Identification of X and V growth factor requirements 

 

H. influenzae is a fastidious organism that can be distinguished from other strains of Haemophilus on 
the basis of its growth requirements for media containing haemin (X factor) and nicotinamide 
adenine dinucleotide (NAD, V factor). H. hemolyticus is the only other species requiring X and V 
factors, and can be distinguished from H. influenzae by looking for hemolysis on a blood agar plate. 

The standard medium for growth is chocolate agar, because the processing of heating the blood 
during preparation makes both X and V factors available to the organism. IEIP will supply 
supplemented, commercially prepared chocolate agar plates. 

 

1) Prepare a heavy suspension of cells (No. 1 McFarland) from a primary isolation plate in a 
suitable broth (tryptone-based soy broth or TSB, or heart infusion broth). If the primary 
isolation plate contains insufficient growth or is contaminated, make a subculture on a 
chocolate agar plate. When preparing the media, avoid transfer of agar medium to the 
broth; even the smallest sample of agar will affect the test and may lead to 
misidentification of the bacteria because the agar contains X and V factors. 



2) Inoculate a heart infusion or tryptone-based soy agar plate by streaking a sterile swab of 
the suspension over one-half of the plate in at least two directions to ensure confluent 
growth. Allow the plate to dry. 



3) Using sterile forceps, place paper strips or disks containing X, V, and XV factors on the 
inoculated plate spaced equally far apart from each other and press gently against the 
agar. 



4) Incubate the plates in an inverted position (upside down) at 35C in a 5% CO2 incubator 
for 18-24 hours. 



5) H. influenzae will grow only around the XV disk (i.e., the disk containing both X and V 
factors). 





Species 

X and V Factor Requirements 

-hemolysis 

on blood agar 

X 

V 

H. influenzae 

+ 

+ 

- 

H. parainfluenzae 

- 

+ 

- 

H. hemolyticus 

+ 

+ 

+ 

H. parahemolyticus 

- 

+ 

+ 

H. aphrophilus 

+ 

- 

- 

H. paraphrophilus 

- 

+ 

- 



 


Identification of the H. influenzae serotype

 

H. influenzae is currently recognized to have 6 serotypes (a, b, c, d, e, and f). H. influenzae type b (Hib) 
is a major cause of invasive bacterial disease, and the major cause of meningitis, in unvaccinated 
children in many parts of the world. All Haemophilus isolates should be tested with Hib antiserum, an 
antiserum to one of the other groups, and saline. A strongly positive (3+ or 4+) agglutination 
reaction with type b antiserum and no agglutination with an antiserum to the other serotypes and 
saline is evidence of Hib. All identification testing for H. influenzae serotypes will be conducted at 
the Thai NIH. 

 

Antisera should be stored in the refrigerator at 4C when not in immediate use. Screening an isolate 
first with polyvalent antiserum (which contains antisera to all six recognized serotypes) and a saline 
control will be performed to save resources. If the isolate is positive with a polyvalent antiserum 
and negative in the saline control, technicians will proceed by testing the isolate with type b 
antiserum. If the serotype b reaction is negative, technicians will then test with the remaining type-
specific antisera (a, c, d, e, and f). If an isolate is non-agglutinating in the polyvalent antiserum, the 
assumption will be made that it is either non-typeable, or that it is not H. influenzae and steps will be 
taken to re-confirm the species identity. 

 

Slide agglutination test for serotyping suspected H. influenzae isolates 

 

1) Divide a new, pre-cleaned glass slide into equal sections with a wax pencil or other 
marker. 



2) Collect a small portion of growth from the surface of an overnight culture on chocolate 
agar (without bacitracin), a Haemophilus ID plate, or a Haemophilus test medium plate with 
a sterile inoculating loop. Make a moderately milky suspension of the test culture 
(roughly equivalent to a 6 MacFarland turbidity standard) with 250 l of 0.5% 
formalinized physiological saline and vortex the suspension. 



3) For the agglutination reaction, transfer a loopful (5-10 l) of the cell suspension to the 
lower portion of two sections of the prepared slide. Use enough suspension so that it 
does not dry before testing. 



4) Add 5-10 l of polyvalent antiserum above the drop of suspension in one test section on 
the slide. In an adjacent section of the slide, use the same method to add a 5-10 l drop 
of saline above the final drop of suspension. The loop used in the antiserum must not 
touch the cell suspension or the other antiserum being tested; if it does, it must not be 
placed back into the source bottle of antiserum to prevent contamination of the source 
bottle. 



5) Using a sterile toothpick or sterile loop for each section, mix the antiserum or saline with 
the corresponding drop of cell suspension. Avoid contamination across sections of the 
slide. 



6) Gently rock the slide back and forth for up to 1 minute, being careful to avoid motions 
that cause the mixtures to run together. After one minute of rocking, observe the mixed 
drops and read the slide agglutination reactions under a bright light against a black 
background. 



7) In a strong reaction, all the bacterial cells will clump and the suspension fluid will appear 
clear. Only strong agglutination reactions (3+ or 4+) are read as positive. If strong 
agglutination occurs in the polyvalent antiserum, use the same methods to test the isolate 
with type b and other type-specific antisera. If agglutination does not occur, continue 
testing the isolate to confirm whether it belongs to another Haemophilus species. 


 




Antimicrobial susceptibility testing of H. influenzae 

 

Antimicrobial susceptibility testing for isolates of H. influenzae will be performed as described above 
for S. pneumoniae in Section 6.6, with the following exception: 

 

 The recommended medium for antimicrobial susceptibility testing for H. influenzae is 
Haemophilus test medium (HTM), which will be prepared by the Thai NIH and supplied to 
the provincial laboratories by IEIP. The zone diameter sizes can be interpreted correctly 
only when HTM is used, as per NCCLS standards. 



 The 10 g ampicillin disk predicts both intrinsic (penicillin-binding protein-mediated) and -
lactamase-mediated penicillin and ampicillin resistance and should be used when testing H. 
influenzae. 







___AcroPDFMTS

6.9 Shipping clinically significant isolates for further testing 

 

Any clinically significant isolates obtained through patient hemocultures should be shipped to the 
IEIP laboratory in Nonthaburi (as described in Section 4.3), which will forward them to the Thai 
NIH for long-term storage at -70C and/or further testing (see flow chart below). 

 

Careful disinfection of the patients skin, the nurse or laboratory technicians gloves, and the surface 
of the blood culture bottles prior to inoculation and incubation of the blood culture bottles can 
reduce the numbers of contaminated blood cultures. Despite these precautions, some proportion of 
inoculated blood cultures will yield positive growth of bacteria commonly found on the skin or in 
the environment and rarely (if ever) associated with serious diseases. 

 

No absolutely conclusive method exists to differentiate a contaminant from a true bacteremia, 
although comparing multiple blood cultures from a single patient (a technique employed in the U.S., 
but not recommended by this protocol) can help. Even organisms known as common skin 
contaminants can cause disease in severely immunocompromised patients, particularly in those with 
indwelling central venous catheters. The final conclusion on whether an isolate represents a 
clinically significant pathogen or a culture contaminant must be made by the clinician who 
ordered the blood culture, on the basis of the laboratory and clinical information available. 

 

However, the laboratory microbiologist can use some existing tools to determine the likelihood that 
a microorganism represents a true bacteremia rather than a blood culture contaminant:25,26,27

 

1. The isolation of virulent human pathogens such as S. pneumoniae, Staphylococcus aureus, 
Klebsiella species, Pseudomonas species, E. coli and other Enterobacteriacae species, H. 
influenzae, or N. meningitides from blood culture generally indicates true bacteremia. 



2. In contrast, the following suggest the possibility of contaminated cultures: 
a. Prolonged incubation before the culture becomes alarm-positive; 
b. Multiple organisms from a single culture (unless the patient is severely 
immunocompromised); 
c. The organism isolated appears inconsistent with the patients clinical condition; 
d. Growth yields bacteria that frequently reside on the skin (such as Corynebacterium 
species, Bacillus species, or Propionibacterium acnes) or in the local environment (such as 
Brevibacterium species in an area with many dairy farms) and rarely cause human 
disease. Although coagulase-negative staphylococci usually represent contaminants, 
up to 15% of isolates may be true bacteremias; this is more likely in patients with 
indwelling catheters or other implanted devices. 







The laboratory microbiologist will have to decide whether isolates are as likely as not to represent 
clinically significant isolates when shipping these to IEIP for preservation and possible further 
testing at the Thai NIH. All isolates of S. pneumoniae should be assumed to be clinically significant.. 

 

Preparing isolates for shipping

 

Because of the particular focus on S. pneumoniae and, to a lesser extent, H. influenzae isolates, Dorset 
Egg media slants have been selected as a standard storage and transport medium to increase the 
chances of preserving these two fastidious organisms. Dorset Egg medium slants will be prepared 
by the Thai NIH and supplied by IEIP to the provincial laboratories monthly. 


1) Using a sterile inoculating wire loop, touch a colony from a freshly prepared overnight 
culture of the isolate (as a pure culture grown on blood agar at 35C with 5% CO2). 



2) Inoculate a 4 mL Dorset Egg media slant by streaking the isolate onto the surface and 
store at room temperature (18-25C). 



3) Repeat with a second colony from the same plate and a second slant. 



4) With a sterile inoculating wire, remove another colony from the freshly prepared 
overnight culture and plunge the wire into the center of the prepared nutrient agar tube. 



5) Repeat with another colony and a second nutrient agar tube. 



6) Ship all isolates to IEIP every Tuesday at room temperature with the provided shipping 
list. 





 

 


Inoculated hemoculture bottles

Positive alarm

No bacteria 
isolated 

No organisms on 
Gram stain or solid 
media 

Bacteria isolated

Gram stained 

Cultured on solid 
media 

Biochemical testing 

 

Cannot confirm 
identity

Bacteria isolated 

Gram stained 

Cultured on solid 
media 

Biochemical testing 

 

Confirmed identity 

Clinically significant 

Ship to IEIP/Thai NIH 

- Cryopreserve isolates 

- Confirmatory testing (QC) 

- Serotyping and penicillin Etest 

Ship to IEIP/Thai NIH

- Identification 

- Antimicrobial 
susceptibility testing

Test for autolyzed 

pneumococcus 

Use modified Binax 
NOW protocol 

Ship culture bottle to 
IEIP/Thai NIH 


Appendix 1: 

Quality Control and Quality Assessment 

 

A stringent QC/QA program relies upon consistent inclusion of appropriate reference strains in 
standard microbiology testing; systematic monitoring of environmental conditions and equipment; 
careful record-keeping; and regular training and refinement of laboratory protocols. Throughout these 
Standard Operating Procedures, specific actions to establish the quality of media, reagents, conditions, 
and testing procedures have been described. The list below provides a brief overview of critical 
components of general QC testing. 

 

In order to demonstrate that blood culture process implemented in the provinces of Nakhon Phanom 
and Sa Kaeo can be trusted by clinicians and patients, IEIP will support the provincial hospital 
microbiology laboratories in establishing a rigorous QC/QA system. 

 

 The IEIP laboratory microbiologist will oversee the routine performance of daily and weekly 
internal QC procedures; 
 IEIP staff will visit each provincial microbiology laboratory monthly to review the records 
documenting internal QC procedures, to discuss any modifications necessary to improve 
protocols, and to arrange regular training sessions as indicated; 
 IEIP staff will also conduct a quarterly external QA audit of each provincial microbiology 
laboratory, including a review of record-keeping and data entry, and introduction of blinded 
test samples into the blood culture system in the form of spiked control bottles. Several 
national and international external quality assessment schemes will be considered as an 
additional layer of monitoring; the laboratories at Crown Prince Hospital in Sa Kaeo have 
already contracted with a Thai MOPH-sponsored QA auditing program, and plan to use this 
program as an external check for at least the first year of the project. 





The development of criteria for success will be an ongoing collaboration between the laboratory 
personnel on-site in the district and provincial hospitals, and those at IEIPs Bangkok facilities. 
Nonetheless, it is expected that laboratories will show a consistent trend toward improvement in 
conducting internal QC testing and meeting external QA challenges. 

 


Expected accomplishments in QC/QA testing 

 

Action/Milestone 

Year 1 

Year 2 

Year 3 

Documentation of daily and weekly 
QC monitoring, including: 

 environmental conditions 
 equipment function 


90% complete 

95% complete 

99% complete 

Documentation of all steps in 
specimen processing, including: 

 transport temperatures 
 specimen volume 
 specimen rejection forms 


90% complete 

95% complete 

100% complete 

Documentation of inclusion of 
standard reference strains as 
appropriate (weekly, daily, or with 
each test) for all: 

 inoculation of solid media 
 antimicrobial susceptibility 
 biochemical tests 
 biological tests 


 

85% complete 

 

90% complete 

 

95% complete 

Accuracy and completeness of 
record-keeping and information-
sharing, including: 

 Completion of Specimen 
Collection Forms in real 
time, with related forms 
attached 
 Prompt placement of all 
forms in patient charts 
 Prompt notification of 
clinicians (as recorded and 
through clinician feedback) 
 Completion of bound 
logbooks in real time 





90% complete 

95% complete 

99% complete 




Maintenance of the GAVI 
Microbiology Database (as 
determined by quarterly audit): 

 Accuracy (correspondence 
of Specimen Collection 
Forms, laboratory logbooks, 
blood culture system 
Observa software, and data 
entered through Access) 
 Completeness 
 Timeliness (completed 
records entered the first time 
within one week of 
recording results in lab, and 
entered the second time 
within two weeks after 
receipt at IEIP offices) 


90% 

95% 

99% 

Successful identification and 
characterization of antimicrobial 
susceptibility of a panel of 5-10 
unknown organisms, introduced as 
blinded specimens as part of an 
quarterly external QA review 

 

80% 

 

85% 

 

95% 



 

 

 

I.i Recording information on routine internal QC testing 

 

All QC testing should be documented by recording the results, data, and time in the appropriate 
format. For daily evaluation of equipment and environmental conditions, record sheets (based on the 
templates provided by Boonchuay Eampokalap, Chief of Microbiology, Bamrasnaradura Institute of 
Bamrasnaradura Hospital, Thai Ministry of Public Health) should be kept with the instrument or in a 
nearby, easily accessible location until complete, and then collected in a binder. Personnel should be 
assigned to complete records on equipment and environmental conditions on a regular basis. 
Malfunctioning equipment should be reported first to the IEIP microbiologist in each laboratory, with 
arrangements for repairs referred through the provincial Surveillance and Research Coordinators 
office. The IEIP microbiologists will also be responsible for establishing a plan for regular 
maintenance (most frequently annual) for all equipment, and making logistical arrangements through 
the Surveillance and Research Coordinators office. 

 

For results obtained using standard reference strains in evaluating media and growth conditions, 
antimicrobial susceptibility testing, and biochemical testing, results should be recorded in a bound 
laboratory logbook, including the date and time. Instructions on performing appropriate QC testing 
should be kept in the front of each logbook for easy reference. 


 

Equipment/test/condition 

Action 

Performed

Equipment 

 

 

 

BacT/ALERT 3D automated 
blood culture system 

Evaluate whether temperatures are below, within, 
or above range (using internal thermometer)  
calibrate or request technical assistance if 
necessary. Move samples to back-up instrument if 
possible until problem is solved. 

Daily 

(2-3 times) 

 

All incubators 

 

Refrigerators and freezers 

CO2 incubators 

Check CO2 levels in incubator and pressure 
remaining within tank  adjust or request technical 
assistance if necessary. Have candle-jars on hand 
as back-up if only one CO2 incubator is available. 

Daily 

(2-3 times) 

Power supply 

Note all fluctuations and which 
samples/equipment might have been affected. 

As 
necessary 

Autoclaves 

Sterilization testing (tape indicator). 

With each 
use 

Autoclaves 

Biological testing (spore test) 

Weekly 

Biosafety cabinet 

Check air-flow and gauges for air pressure 

Daily (2-3 
times) 

Conditions 

 

 

Room temperature 

Check to see if temperature is below 15C or 
exceeds 30C; this could affect the function of the 
blood culture machine and incubators. Take action 
to decrease temperatures (closing blinds, increasing 
air-conditioning) if necessary. Do not turn off air-
conditioning at night. 

Daily 

(2-3 times) 

Temperature during shipping 
of inoculated blood cultures 

Check TempTale device in each insulated box 
upon receipt of specimens, rejecting any specimens 
that have been too long out of temperature range 
(18-30C  see Section 4). 

With each 
drivers 
shipment 




 

Test 

 

 

Culturing isolates on solid 
media 

Streak appropriate standard reference strain for 
each media plate. 

Weekly, 
and with 
each new 
batch of 
media 

Antimicrobial susceptibility 
testing 

Test a susceptible standard reference strain for the 
isolate using the same media and antibiotic disks 
(for example, an S. pneumoniae isolate should be 
compared to an S. pneumoniae standard reference 
strain). 

Every day 
up to 30 
consecutive 
days of in-
range 
results, 
then 
weekly and 
with each 
new batch 
of media or 
disks 

Biochemical tests 

Include an appropriate standard reference strain 
for the test (such as a bile soluble S. pneumoniae 
standard reference strain for the bile solubility test). 

With every 
test 

Gram and AFB staining 

Include one slide with an appropriate positive and 
negative standard control strain for each type of 
staining. 

Weekly, 
and with 
each new 
staining kit 



 

 

I.ii Selecting appropriate standard reference strains 

 

For each batch of media, biological, or biochemical assay, standard reference strains should be tested 
in parallel with strains isolated from patient blood cultures, and the results compared to published 
observations. Failure of the standard reference strains to perform as expected should prompt a 
review of the techniques used, the environmental conditions, equipment function, and media or 
reagent quality until the problem can be identified and corrected. 


 

Organism 

Assay 

Characteristic 

Streptococcus pneumoniae 

ATCC 49619 

 

Growth on blood agar 

 

Antimicrobial susceptibility testing 

 

Optochin test 

 

Bile solubility test 

Growth positive (a-hemolysis) 

 

NCCLS standard reference strain 

 

Optochin sensitive 

 

Bile soluble 

Streptococcus pyogenes 

ATCC 19615 

Growth on blood agar 

 

Catalase test 

Growth positive (-hemolysis) 

 

Catalase negative 

Streptococcus bovis 
(gallolyticus) 

ATCC 49147 

Optochin test 

 

Bile solubility test 

Optochin resistant 

Non-bile soluble 

Staphylococcus aureus 

ATCC 25923 

Growth on blood agar 

 

Coagulase test 

 

Catalase test 

 

Gram staining 

 

Antimicrobial susceptibility testing 

Growth positive (.-hemolysis) 

 

Coagulase positive 

 

Catalase positive 

 

Gram-positive 

 

NCCLS standard reference strain

Staphylococcus epidermidis 

ATCC 12228 

Coagulase test 

Coagulase negative 

Haemophilus influenzae 

ATCC 49247 


Growth on chocolate agar 

 

X and V Factor requirements 

 

Antimicrobial susceptibility testing 

Growth positive 

X and V Factor dependent 

NCCLS standard reference strain

Haemophilus influenzae 
ATCC 49766 

Antimicrobial susceptibility testing 

Sensitive strain for some 
antibiotics 

Neisseria meningitidis 

ATCC 13077 

Growth on chocolate agar 

 

Oxidase test 

 

Carbohydrate utilization test 

Growth positive 

 

Oxidase positive 

 

Glucose +/maltose + 




 

Organism 

Assay 

Characteristic 

E. coli 

ATCC 25922 

Growth on MacConkey agar 

 

Growth on blood agar 

 

Triple Sugar Iron Agar (TSI) 

 

Oxidase test 

 

Gram staining 

 

Urease agar test 

 

Indole spot test 

 

Growth on citrate agar 

 

Lysine iron agar 

 

Antimicrobial susceptibility testing 

Growth positive (pink colonies) 

 

Growth positive (no hemolysis) 

 

Acid butt/acid slant/gas +/H2S + 

 

Oxidase negative 

 

Gram negative 

 

Urease negative 

 

Indole positive 

 

Growth negative 

 

Acid butt/alkaline slant/H2S - 

 

NCCLS standard reference strain 

Pseudomonas aeruginosa 
ATCC 27853 

Oxidase test 

 

Indole spot test 

 

Antimicrobial susceptibility testing 

Oxidase positive 

 

Indole negative 

 

NCCLS standard reference strain 

Salmonella typhimurium 

ATCC 14028 

Growth on MacConkey agar 

 

 

Growth on citrate agar 

 

Lysine iron agar 

 

Triple Sugar Iron Agar 

 

Growth positive (colorless 
colonies) 

 

Growth positive 

 

Alkaline butt/alkaline slant/H2S + 

 

Acid butt/alkaline slant/gas 
+/H2S + 

Klebsiella pneumoniae 

ATCC 35657 

Urease agar test 

Urease positive 



 


I.iii Preserving reference strains for routine laboratory use28

 

While many methods exist for preserving reference strains for long periods of time, the bead stock 
method has many advantages for laboratories with limited resources and freezer space. Bacteria 
stick to the charged beads, allowing retrieval of and quick inoculation of solid media with the 
preserved strains. Isolates stored at -70C or below may be stored indefinitely without losing 
viability; storage at -20C is not recommended. IEIP will supply glycerol for the freezing medium, 
cryovials, and beads, as well as the standard reference strains. 

Freezing reference strains as bead stocks 

1) Stocks should be prepared in biological safety cabinet. 
2) Inoculate a non-selective solid media slant (such as tryptone-based soy agar, TSB, or 
brain-heart infusion agar, BHIB) and incubate overnight (18-24 hours) at 35C. 
3) Harvest the freshly grown bacteria from the surface of the slant with a sterile 
bacteriological loop and suspend in freezing medium (TSB or BHIB plus 15% glycerol). 
4) Label cryovials with the name of the reference strain and the date, and add 20-30 beads 
to each cryovial. 
5) Dispense the bacterial suspension into the cryovials, making sure that the beads are fully 
submerged in the suspension. 
6) Carefully remove the excess suspension, pouring away as much of the suspension as 
practical without spilling the beads. Seal the cryovial with the included cap. 
7) Place dry ice in a leakproof metal container large enough to hold a metal culture rack, 
and add enough ethyl alcohol to submerge about half of the cryovial. If dry ice cannot 
be obtained, place a container of the alcohol in the -70C freezer overnight. 
8) Freeze the suspension rapidly by placing the sealed vials in the dry ice bath. Transfer the 
vials to a -70C freezer. 





Recovery of cultures from frozen storage 

 

1) Place frozen cultures on dry ice or into an alcohol and dry ice bath and transfer to a 
biological safety cabinet. 
2) Using a sterile loop or forceps, remove a bead from the cryovial, being careful not to 
contaminate the top or inside of the vial. Roll gently on an appropriate solid media plate 
to inoculate, then streak to isolation. 
3) Reseal and return vial to freezer before the contents completely thaw. By maintaining 
the temperature of the bead stock and using aseptic technique, transfers can be made 
from the same vial multiple times. 


Working reference strain stocks 

1) Select a single isolated colony from the newly prepared solid media plate (above). 



 


2) Prepare a working culture by inoculating a non-selective solid media slant (such as TSB) 
and incubate overnight (18-24 hours) at 35C. (Use a chocolate agar slant for H. 
influenzae and Neisseria strains.) 
3) Store the working culture slant at 4C. Subculture QC testing cultures from the working 
culture slants prior to schedule testing (i.e., daily or weekly). 
4) Discard the working culture slants after one month and replace from reference strain 
stocks stored at -70C. 





I.iv Antimicrobial susceptibility test breakpoints and quality control ranges 

 

For the expected results and testing ranges for antimicrobial susceptibility testing, please consult the 
most recent version of the Clinical and Laboratory Standards Institute (formerly NCCLS) 
Performance Standards for Antimicrobial Susceptibility Testing and the Manual for Identification and 
Antimicrobial Susceptibility Testing. Both will be supplied to the provincial hospital microbiology 
laboratories by IEIP. 


Appendix 2: 

Testing Patient Serum for 

Pre-hospital Antibiotic Use 

 

 

Patient use of antibiotics can affect the sensitivity of any blood culture system. The impact of pre-
hospital antibiotic use by patients in rural Thai hospitals (obtained through private clinics or by over-
the-counter purchases) is difficult to predict. Therefore, disks soaked in serum will be prepared for 
each patient receiving a blood culture as ordered by a Thai clinician, and cryopreserved until a 
convenient time for later testing. A strain of Staphylococcus highly susceptible to many commonly 
used antibiotics will be used to determine qualitatively whether the serum contained antibiotics at 
the time of the blood draw. The following method will be used to test patient sera for pre-hospital 
antibiotic use: 

 

1) Suspend viable colonies from a fresh pure culture of Staphylococcus aureus ATCC 9144 (NCTC 
6571) grown overnight on nutrient agar in TSB or sterile physiological saline. 



2) Adjust the cell suspension to the density of a 0.5 McFarland turbidity standard (described in 
Appendix 3), carefully avoiding froth or bubbles while mixing. Compare the density of the 
suspension to the 0.5 McFarland turbidity standard by holding the suspension and the 
McFarland standard in front of a light against a white background with black lines. Add 
more saline or broth if the density appears too heavy, and more bacteria if the density 
appears too light. 



3) Dip a sterile cotton swab into the bacteria suspension, removing excess fluid by rotating 
against the sides of the dilution tube. 



4) Use the same swab (without re-dipping in the bacterial cell suspension) to inoculate the 
entire surface of a nutrient agar plate three times, rotating the plate 60 degrees between each 
inoculation to achieve even coverage. 



5) Allow the inoculum to dry before placing disks on the plates (5-15 minutes). 



6) Remove the patient serum disks from the -20C or -70C freezer and allow to warm to room 
temperature, thawing only the disks to be used that day. Label each plate appropriately. 
Disks from 3-4 patients can be placed on each plate as long as sufficient space is allowed and 
they are labeled clearly. 



7) Place each patient serum disk on a plate using sterile forceps, tapping it gently to ensure that 
it adheres to the agar. Once a disk contacts the agar surface, diffusion of any drugs may 
begin immediately, and the disk should not be moved. 



8) Prepare one plate using commercially available antibiotic susceptibility testing disks. Include 
one untreated paper filter disk soaked only in sterile saline as a negative control. 






9) Incubate the plates in an inverted position (upsidedown) in a 35C incubator overnight (18-
24 hours). 



10) Examine each patient serum disk for a zone of inhibition indicating antimicrobial activity. 
Measure the diameter of the zone of inhibition with a rule or calipers, measuring the distance 
from the last visible colony on each side of the disk from the top surface of the plate with 
the lid removed. Care should be taken not to touch the disk or surface of the agar with the 
ruler or calipers. 



11) Confirm that results for the positive (commercial antibiotic) and negative disks lie within 
acceptable standards. 



12) Record the diameter of the zone of inhibition around each patient serum disk on the original 
Specimen Collection Forms. The running number from the cryotube should be used to 
record results so that these can be tied to the blood culture results. Because the antibiotic 
used may not be known (precluding comparison to a control), the presence of a zone of 
inhibition will be classified as suggestive of, not suggestive of, or inconclusive for pre-
hospital antibiotic used. 



Appendix 3: 

Preparing Media and Reagents 

 

IEIP will provide all solid media for confirmatory identification and testing, prepared by either a 
commercial manufacturer (CLINAG, Bangkok, Thailand) or by the Thai National Institutes of Health 
(Nonthaburi, Thailand). However, laboratory technicians must still include appropriate standard 
reference strains to test media quality every time a new batch of media is used. Standard protocols for 
preparing commonly used microbiological media are also included below for reference and for use by 
the provincial laboratories in preparing supplemental media, or media for uses other than the IEIP 
Microbiology Project. 

 

III.i Considerations for quality control of media 

 

Each batch of medium should be tested for the ability to support growth of the target organisms as 
appropriate, in the laboratory conditions within the provincial microbiology laboratories. An 
appropriate standard reference strain (listed in Appendix 1, and described below specifically for each 
type of common media) should be inoculated onto each type of media plate one per week, and every 
time a new batch of medium is used. 

 

For selective media, use at least one organism that will grow on the medium and one that will not 
grow to test the mediums ability to differentiate between the two. If the medium is also differential, 
include two organisms that will grow on the medium and produce different reactions. 

 

Methods for quality control of media

 

1) Inoculate the control strain to non-selective broth (such as tryptone-based soy broth, or 
TSB) and incubate overnight at 35C. 



2) Prepare a standardized inoculum for testing the medium: 



a. If testing selective or inhibitory media, dilute the overnight broth culture 1:10 with 
sterile broth; 
b. If testing nonselective media, dilute the overnight broth culture 1:100 with sterile 
broth. 





3) Inoculate one plate (or tube) of each medium to be tested with 100 L of the diluted 
control strain, and streak to isolation. 



4) Incubate at 35C overnight (18-24 hours) under appropriate CO2 conditions for the 
media and organism. 


 





III.ii Preparation and QC testing of commonly used microbiological media 

 

Routine Agar and Broth Media 

 

All agar media can be dispensed into 15x100-mm Petri dishes (15 to 20 ml per dish). After pouring, 
leave the plates at room temperature for several hours to prevent excess condensation from forming 
on the covers of the dishes. Store the plates in a secured plastic bag that does not allow movement 
of plates in an inverted position at 4C  a re-sealable bag is not an appropriate container, as plates 
can slide and become uncovered in storage. 

 

 

Heart Infusion Agar (HIA) and Trypticase Soy Agar (TSA) 

 

These media are general purpose media used with or without blood for isolating and cultivating a 
number of microorganisms. The media should appear straw colored (a yellowish to gold colouring). 
HIA and TSA are also used for determining the X- and V-factor requirements of H. influenzae. Each 
freshly prepared or purchased batch of HIA or TSA should be quality control-tested by determining 
the X and V requirements of H. influenzae. A fresh plate is inoculated with a control strain; X, V, and 
XV disks should be placed on the inoculated plate. H. influenzae should grow only around the XV 
disk. 

 

1) Prepare the volume of HIA or TSA according to the instructions on the label of the 
dehydrated medium in flasks. Media should be fully dissolved with no powder on the 
walls of the vessel before autoclaving. 



2) Autoclave at 121C for 20 minutes. 



3) Cool to 50C and pour into 15x100-mm Petri dishes. Allow to solidify and lids to dry 
before storing in plastic bags at 4C until used. 


 




Blood Agar Plate (BAP): TSA + 5% Sheep Blood 

 

BAP is used as a general blood agar medium (sheep blood agar plate) for most testing purposes. It 
is also used for the optochin test and subcultures of S. pneumoniae. (Human blood agar should never 
be used for the isolation or testing of cultured clinical specimens such as S. pneumoniae as part of the 
blood culture process, as natural and pharmaceutical antibiotic factors in human blood can affect the 
growth of isolates. Only high-quality animal blood should be used; IEIP can provide further 
guidance on availability as necessary.) The plates should appear a bright red color. A dark red 
appearance indicates that the blood was added when the agar was too hot. If this happens, discard 
the medium and prepare another batch. Test each new, freshly prepared or purchased batch of BAP 
for growth and haemolytic reaction with a strain of S. pneumoniae. The colonies are small and should 
appear grey to grey-green surrounded by a distinct greenish halo in the agar. 

 

1) Prepare TSA according to the instructions given on the label of the dehydrated powder, 
adding 20 g of agar to 500 mL water in a l-litre flask. Heat to dissolve. 



2) Autoclave at 121C for 20 minutes. Cool to 50C. 



3) Add 5% sterile, defibrinated sheep blood (25 ml sheep blood per 500 ml agar  adjust 
accordingly for the total volume). 



4) Dispense 20 ml into 15x100-mm Petri dishes. Allow to solidify and dry, then store in a 
plastic bag at 4C. 


 




Heart Infusion Rabbit Blood Agar Plate (HIA - Rabbit Blood) 

 

HIA-rabbit blood is used for determining the hemolytic reaction of Haemophilus species. This 
medium should appear bright red and look very similar to BAP. If the medium is dark red, discard 
and prepare a new batch. Horse blood may be substituted for rabbit blood in this medium. A strain 
of H. haemolyticus should be used to quality control the proper growth and 

haemolytic reactions of the medium. H. haemolyticus should grow well and be surrounded by a 
distinct zone of complete haemolysis which appears as a clear halo surrounding the colonies. 

 

1) Prepare the HIA according to the instructions on the label of the dehydrated medium. 



2) Autoclave at 121C for 20 minutes. Cool to 50C in a water bath. 



3) Add 5% sterile, defibrinated rabbit blood (5 ml/100 ml of medium) and dispense into 
15x100-mm Petri dishes. Allow to solidify and dry for a few hours before storing in a 
plastic bag at 4C. 


 




Trypticase Soy Broth (TSB) 

 

TSB is a non-selective broth used for making suspensions of H. influenzae prior to testing for X and 
V- factor requirements. Heart infusion broth, sterile saline or phosphate-buffered saline may be 
substituted for TSB. H. influenzae does not grow in TSB but the medium should not be toxic to other 
bacteria. Therefore, S. pneumoniae should be used to quality control for toxicity. Inoculate a tube of 
medium with a loop of freshly growing strain of S. pneumoniae; incubate overnight at 35C. The 
broth should be turbid the next day. Subculture the broth to test for proper growth characteristics of 
S. pneumoniae (use a BAP). 

 

1) Prepare the TSB according to the instructions on the label of the dehydrated medium. 



2) Dispense 5 ml into 15x125-mm tubes, autoclave at 121C for 20 minutes, cool, and store 
at 4 C. 


 




Chocolate Agar Plate (CAP) with TSA and Growth Supplement 

 

CAP with growth supplements is a medium that supports the special growth requirements needed 
for the isolation of fastidious organisms, such as H. influenzae and N. meningitidis, when incubated in a 
5% CO2 atmosphere. CAP has a reduced concentration of agar, which increases the moisture 
content of the medium. Supplemented chocolate agar should support the growth of N. meningitidis, 
H. influenzae, and S. pneumoniae. Chocolate agar slants for transport and short-term storage can be 


prepared in the same manner as that described for agar plates, except that the medium is dispensed 
in 16x125 mm screw-cap tubes and slanted before solidifying. All freshly prepared or purchased 
chocolate agar media should be tested with the three species of bacteria to determine the mediums 
capacity to support their growth. 

 

Chocolate agar slants should look brown to brownish-red in colour. N. meningitidis and H. influenzae 
should appear as a greyish, almost translucent film on the slants surface with no discoloration of the 
medium after 24 hours of incubation. S. pneumoniae should appear as small grey to grey-green 
colonies with a very distinct greenish discoloring of the medium. If the medium does not support 
the growth of one or all of the bacteria, discard and prepare or purchase a fresh batch. If H. 
influenzae does not grow, the IsoVitaleX or its equivalent may have been inadvertently omitted. 

 

1) Using TSA as the basal medium. Prepare double strength (20 g in 250 ml distilled water). 



2) Autoclave, and cool to 50C. Use the thermometer to verify the cooling temperature. 



3) Hemoglobin solution: Prepare a solution of 2% hemoglobin (5 g in 250 ml distilled 
water). Mix the hemoglobin in 5-6 ml distilled water to form a smooth paste. Continue 
mixing as the rest of the water is added. Autoclave, and cool to 50C. 



4) Add the hemoglobin solution to the double-strength TSA and continue to hold at 50C. 



5) If a hemoglobin solution is unavailable, an alternative is to add 5% sterile defibrinated 
sheep, rabbit, guinea pig, or horse blood (5 ml blood per 100 ml agar) to full-strength 
TSA agar base (20 g in 500 ml distilled water). DO NOT use human blood. After the 
base medium has been autoclaved and cooled to 50C, add the blood and place in a hot 
water bath at no more than 80C for 15 minutes or until a chocolate color is achieved. 
Then cool to 50C. 



6) After the hemoglobin solution or the defibrinated blood has been added to the base 
medium and the medium has cooled to 50C, add growth supplement (such as 
IsoVitaleX or Vitox) to a final concentration of 1%. Mix the ingredients by gently 
swirling the flask; avoid forming bubbles. 



7) Dispense 15 to 20 ml in each 15x100 mm Petri dish. 


 




Cystine Trypticase Agar (CTA) with 1% Carbohydrate 

 

1) Suspend 28 g of CTA medium or 29.5 g in 900 ml of distilled water. Mix thoroughly, 
heat with frequent agitation, and bring to a boil for 1 minute to completely dissolve the 
powder. 



2) Autoclave the flask at 121C for 15 minutes. Cool to 50C 



3) Prepare 10% glucose solution using 10 g glucose in 100 ml of distilled water. Filter 
sterilize using a 0.22 micron filter. 






4) Aseptically add this entire solution (100 ml of 10% glucose solution) to the 900 ml of the 
CTA medium to obtain a final 1% concentration of the glucose. 



5) Dispense 7 ml in 16 x 125 mm glass tubes with screw caps and store at 4C. 



6) Repeat this procedure for the remaining 3 carbohydrates: maltose, lactose and sucrose. 


 




III.iii Preparation of commonly used microbiological reagents 

 

Reagents for Gram Stain (Hucker Modification) 

 

Ammonium oxalate-crystal violet: 

 

1) Solution a: Dissolve 2.0 g crystal violet (certified) in 20 mL 95% ethyl alcohol. 



2) Solution b: Dissolve 0.8 g ammonium oxalate in 80 mL distilled water 80.0 ml. 



3) Mix solutions a. and b. Let stand overnight. Filter through coarse filter paper before use. 





Grams iodine (protect solution from light) 

 

Dissolve 1 g iodine (crystalline) and 2 g potassium in 300 mL distilled water. Grinding the 
dry chemicals in a mortar with small additions of distilled water may be helpful in preparing 
the solution. 

 

Decolorizer 

 

95% ethyl alcohol 

 

Counterstain (safranin) 

 

1) Stock solution: dissolve 2.5 g safranin-O (certified) in 100 mL 95% ethyl alcohol. 



2) Working solution: dilute 10 mL safranin stock solution in 90 mL distilled water. 


 




McFarland Turbidity Standards 

 

Commercially prepared 0.5 MacFarland turbidity standards are available from various manufacturers. 
Alternatively, the 0.5 MacFarland turbidity standard can be prepared by: 

 

1) Add 0.5 mL of a 1.175% (weight/volume, or wt/vol) barium chloride dihydate 
(BaCl2.2H2O) solution to 99.5 mL of 1% (vol/vol) sulfuric acide (H2S04). 



2) Aliquot the turbidity standard into test tubes identical to those that will be used to 
prepare bacterial suspensions. 






3) Mark the level of the liquid, and then seal the McFarland turbidity standard tubes with 
wax, Parafilm, or other seal that can prevent evaporation. 



4) Check the accuracy of the density with a spectrophotometer with a 1-cm light path if 
possible. For the 0.5 McFarland turbidity standard, the absorbance at a wavelength of 
625 nm should be 0.08-0.1. Alternatively, the accuracy of the McFarland turbidity 
standard can be verified by adjusting a suspension of a control strain (such as E. coli 
ATCC 25922) to the same turbidity, preparing serial 10-fold dilutions, and then 
performing plate counts of colonies. The adjusted suspension should give a count of 
108 colony-forming units/mL. 



5) Sealed McFarland turbidity standards may be stored for up to 6 months in the dark at 
room temperature (22-25C). Discard after 6 months, or sooner if the level of liquid in 
the tube drops below the marked level. 



6) Before each use, shake the tube containing the turbidity standard well to mix the fine 
white precipitate thoroughly. 


 

 





QC Record:BLOOD AGAR PLATESRange: Growth/No Growth and HemolysisResultsCommentsNone 
(sterility)*
Growth a-hemolysis Growth -hemolysis Growth .-hemolysis 
21/8/05A-10115/831/10MediaCovvvvvv0/5YExample28/8/05A-10115/831/10MediaCo--vvvv3/5NExampleDate:
WEEKLY QC tests should be repeated for each new batch. Please report out-of-range results to supervisor immediately.
Performed byNameDate 
openedContaminated/
Total plates 
Test: with EACH NEW BATCH 
Reference strain growth/hemolysisPlates*Incubate plates at 35-37?C for 48 hours. If colonies appear, plate is contaminated and sterility testing must be repeated. If more than 1% of plates in batch grow colonies, reject media. 
Number of plates tested (number of plates in batch): 3(=25); 5(26-90); 8(91-280); 13(281-500); 20(501-1200). 
Reviewed by:
Lot numberExpiration 
dateSupplierS. pneumoniae 
ATCC 49619 (1 
plate)
S. pyogenes 
ATCC 19615 (1 
plate)
In expected 
range 
(Y/N)? DateE. coli 
ATCC 25922 (1 
plate) 



QC Record:CHOCOLATE AGAR PLATESRange: Growth/No GrowthResultsDone byCommentsDateLot numberDate 
openedExpiration 
dateSupplierH. influenzae 
ATCC 49247 
Growth 
N. meningitidis 
ATCC 13077 
Growth 
None (sterility) 
Contaminated/
total platesIn 
expected 
range 
(Y/N)?Name21/8/05A-10115/831/10MediaCovv0/5YesExampleDate:
WEEKLY QC tests should be repeated for each new batch. Please report out-of-range results to supervisor immediately.
Test: with EACH NEW BATCH 
Reviewed by:
PlatesReference strain growth 
*Incubate plates at 35-37?C for 48 hours. If colonies appear, plate is contaminated and sterility testing must be repeated. If more than 1% of plates in batch 
grow colonies, reject media. Number of plates tested (number of plates in batch): 3(=25); 5(26-90); 8(91-280); 13(281-500); 20(501-1200). 



QC Record:MACCONKEY AGAR PLATESRange: Growth/No Growth plus Colony ColorResultsDone byCommentsDate21/8/05A-10115/831/10MediaCovvvv0/5YesExampleDate:
Growth 
Reviewed by:
WEEKLY QC tests should be repeated for each new batch. Please report out-of-range results to supervisor immediately.
PlatesDate 
openedLot numberExpiration 
dateSupplierE. coli ATCC 
25922Reference strain growthGrowth 
Pink 
Color*Incubate plates at 35-37?C for 48 hours. If colonies appear, plate is contaminated and sterility testing must be repeated. If more than 1% of plates in batch 
grow colonies, reject media. Number of plates tested (number of plates in batch): 3(=25); 5(26-90); 8(91-280); 13(281-500); 20(501-1200). 
Test: with EACH NEW BATCH 
None (sterility) 
Contaminated/
total platesIn 
expected 
range 
(Y/N)?NameS. typhimurium 
ATCC 14028No 
color


QC Record:GRAM STAINRange: Positive/Negative plus morphology (cocci, bacilli, other)
PositiveNegativeResultsDone byCommentsDateGram stain kit 
(lot number): 
Date 
openedExpiration 
date:
Stains last 
filtered (date):
S. aureus ATCC 
25923E. coli ATCC 
25922In expected 
range 
(Y/N)?Name21/8/05A-10115/6/0531/9/0615/8/05Purple cocciPink bacilliYesExamplePositive = Purple/Negative = PinkTest: WEEKLY and with new stains 
Date:
WEEKLY QC tests should be repeated for new reagents. Please report out-of-range results to supervisor immediately.
Reference strain reactionReviewed by:
Reagents


QC Record:CATALASE TESTRange: Positive/NegativePositiveNegativeResultsPerformed byCommentsDateLot numberPrepared on 
(date)Prepared byS. aureus ATCC 
25923S. pyogenes ATCC 
19615In expected 
range (Y/N)?Name25/8/05A101K. SBubblesNo bubblesYesExampleDate:
Test: with every set of specimens 
Positive = Bubbling/Negative = No bubblingRead: within 15 seconds 
Please report out-of-range results to supervisor immediately.
Reference strain reactionReagentsReviewed by: 



QC Record:OXIDASE TEST (Kovac's method)
Range: Positive/NegativePositiveNegativeResultsPerformed byCommentsDateLot number Date thawedExpiration dateP. aeruginosa 
ATCC 27853E. coli ATCC 
25922In expected 
range (Y/N)?Name21/8/05ABC10115/8/0531/3/06BlueNo changeYExampleDate:Reviewed by:
Please report out-of-range results to supervisor immediately.
Reference strain reactionTest: with every set of specimens 
Positive = Purple/Negative = No colorRead: within 10 secondsReagents 



QC Record:COAGULASE TESTRange: Positive/NegativePositiveNegativeResultsPerformed byCommentsDateLot numberSupplierDate preparedS. aureus ATCC 
25923S. pyogenesATCC 
19615In expected 
range (Y/N)?Name21/8/05ABC101Innovative Res.15/8/05ClotNo clotYExampleDate:
Test: with every set of specimens 
Positive = clotting/Negative = No changeRead: at 4 hours and after 18 hoursPlease report out-of-range results to supervisor immediately.
Reference strain reactionReagents (rabbit plasma)
Reviewed by: 



1 Bartlett JG, Dowell SF, Mandell LA, File TA Jr., Musher DM, Fine MJ. Guidelines from the Infectious Diseases 
Society of America: Practice guidelines for the management of community-acquired pneumonia in adults. Clin Inf Dis 
2000; 31:347-382. 

2 Mandell LA, Bartlett JG, Dowell SF, File TM Jr, Musher DM, Whitney C. Update of practice guidelines for the 
management of community-acquired pneumonia in immunocompetent adults. Clin Infect Dis 2003;37(11):1405-33. 

3 File TM. Community acquired pneumonia. Lancet. 2003 Dec 13;362(9400):1991-2001. 

4 Management of the child with a serious infection or severe malnutrition : guidelines for care at the first-referral level in 
developing countries. World Health Organization, Department of Child and Adolescent Health and Development. 
WHO/FCH/CAH/00.1 (Geneva: 2000). 

5 Adapted from: Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus 
pneumoniae, and Haemophilus influenzae. World Health Organization, Communicable Disease Surveillance and Response. 
WHO/CDS/CSR/EDC/99.7. (Geneva: 2000). 

6 Buttery JP. Blood cultures in newborns and children: optimizing an everyday test. Arch Dis Child Fetal Neonatal Ed 
2002: 87:F25-F28. 

7 Weinstein MP. Blood culture contamination: persisting problems and partial progress. J Clin Microbiol 2003; 41(6)22-
75-78. 

8 Isaacman DJ, Karasic RB, Reynolds EA, Kost SI. Effect of number of blood cultures and volume of blood on 
detection of bacteremia in children. J Pediatr 1996; 128:190-5. 

9 Mermel LA and Maki DG. Detection of bacteremia in adults: consequences of culturing an inadequate volume of 
blood. Ann Intern Med. 1993;119(4):270-2. 

10 Kellogg JA, Manzella JP, Bankert DA. Frequency of low-level bacteremia in children from birth to 15 years of age. J 
Clin Microbiol 2000;38(6):2181-85. 

11 Isaacman DJ, Karasic RB, Reynolds EA, Kost SI. Effect of number of blood cultures and volume of blood on 
detection of bacteremia in children. J Pediatr 1996; 128:190-5. 

12 Little JR, Murray PR, Traynor PS, Spitznagel E. A randomized trial of povidone-iodine compared with iodine tincture 
for venipuncture site disinfection: effects on rates of blood culture contamination. Am J Med. 1999 Aug;107(2):119-25. 

13 Calfee DP and Farr BM. Comparison of Four Antiseptic Preparations for Skin in the Prevention of Contamination of 
Percutaneously Drawn Blood Cultures: a Randomized Trial. J Clin Microbiol. 2002 May; 40(5): 16601665. 

14 Everts RJ, Vinson EN, Adholla PO, Reller LB. Contamination of catheter-drawn blood cultures. J Clin Microbiol 
2001. 39:3393-94. 

15 Technical literature, BacT/ALERT Blood Culture Collection, BioMrieux Inc. (2003). 

16 National Committee for Clinical Laboratory Standards. Procedures for the Collection of Diagnostic Blood 

Specimens by Venipuncture. Approved Standard, H3-A5, Wayne, PA, 2003. 

17 Adapted from: Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus 
pneumoniae, and Haemophilus influenzae. World Health Organization, Communicable Disease Surveillance and Response. 
WHO/CDS/CSR/EDC/99.7. (Geneva: 2000). 

References 

18 Technical literature, BacT/ALERT 3D automated blood culturing machines, BioMrieux Inc.. 

19 Personal communication, technical staff, BioMrieux Inc.. 

20 Berkley JA, Loew BS, Mwangi I, Williams T, Bauni E, Mwarumba S, Ngetsa C, Slack MPE, Njenga S, Hart CA, 
Maitland K, English M, Marsh K, Scott JAG. Bacteremia among children admitted to a rural hospital in Kenya. N Engl 
J Med 2005;352:39-47. 

21 Adapted from Manual for the Laboratory Identification and Antimicrobial Sensitivity Testing of Bacterial Pathogens of Public Health 
Importance in the Developing World. World Health Organization, Centers for Disease Control and Prevention, and U.S. 
Agency for International Development. (Geneva: 2003) and Laboratory methods for the diagnosis of meningitis caused 
by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. World Health Organization, Communicable 
Disease Surveillance and Response. WHO/CDS/CSR/EDC/99.7. (Geneva: 2000). 

22 Adapted from Guidelines for Standard Operating Procedures for Microbiology. World Health Organization, Southeast Asia 
Regional Office. Available at http://w3.whosea.org/microbio/ackf.htm (Geneva: 2001). 

23 Petti CA, Woods CW, Reller LB. Streptococcus penumoniae antigen test using positive blood culture bottles as an 
alternative method to diagnose pneumococcal bacteremia. J Clin Microbiol 2005;43(5)2510-12. 

24 Manual for the Laboratory Identification and Antimicrobial Sensitivity Testing of Bacterial Pathogens of Public Health Importance in the 
Developing World. World Health Organization, Centers for Disease Control and Prevention, and U.S. Agency for 
International Development. (Geneva: 2003) 

25 Weinstein MP. Blood culture contamination: persisting problems and partial progress. J Clin Microbiol 2003; 41(6)22-
75-78. 


26 Richter SS, Beekmann SE, Croco JL, Diekema DJ, Koontz FP, Pfaller MA, Doern GV. Minimizing the workup of 
blood culture contaminants: implementation and evaluation of a laboratory-based algorithm. J Clin Microbiol 2002; 
40(7):2437-44. 

27 Shafazand S and Weinacker AB. Blood cultures in the critical care unit: improving utilization and yield. 
Chest2002;122: 1727-46. 

28 Kay BA. Cryopreservation of reference strains (protocol). U.S. Centers for Disease Control and Prevention, 1995; 
International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), 1986.