# Presentation: 182291

##  Culture Methods 

**C.N. PARAMASIVAN**
**Tuberculosis Research Centre **
**Indian Council of Medical Research **
**Chennai**

## MYCOBACTERIAL  CULTURE

**Advantages**:
**Increases number of cases found**
**Detects cases among smear negative patients**
**Establishes viability  of organisms**
**Distinguishing between Mycobacterial species**
**Helps in performing DST**
**Helps in diagnosing cases of failure**
**Limitations*****:***
**Expensive **
**Require enriched media**
**Require considerable expertise **
**Time consuming**

## SELECTIVE USE OF CULTURE

-   **DRS as a part of N T P evaluation**
**  ****Diagnosis of TB patients with  clinical /radiological  symptom where smears are neg.for AFB**
**  ****Diagnosis of extra pulm.TB/ CH.TB**
**  ****Follow-up of patients who fail on standard regimens to   check up for drug resistance**
**  ****Investigation of high risk individuals who are symptomatic**

## Decontamination Procedures

**1915 – Petroff’s NaOH **
**1946 – Trisodium Phosphate  **
**1955 – Pancreatin Desogen **
**1958 – Pancreatin + 1% cetrimide**
**1962 – Zephiran Trisodium PO****4**
**1963 – N-acetyl L-cysteine + 2%NaOH**
**1969 – Swab culture technique + 1% cetrimide**
**1975 – CPC + NaCl****2**** **

## Culture Media : Solid

**LJ**
**LJ with Na pyruvate**
**LJ with out asparagine**
**Middlebrook’s 7H10 & 7H11**
**Selective 7H10 & 11**
**Ogawa **
**Tarshi’s Blood Agar**

## MODIFIED PETROFF’S METHOD

-  **Used widely in DEDCs since it is  simple, reagents are inexpensive and easy to obtain.**
**NaOH is toxic, both for contaminants and also for tubercle bacilli; therefore, strict adherence to the indicated timings is required**
**Reagents**
**4% sodium hydroxide (NaOH) solution**
**	****	****Sodium hydroxide pellets (analytical grade)****	****4g**
**	****	****Distilled water****	****100ml**
**	****Dissolve NaOH in the distilled water, distribute in conical flasks and sterilize by autoclaving at 121o C for 20 minutes.**
**Sterile distilled water**
**	****	****	****Sterilize by autoclaving in 500ml flasks at 121o C for 20 minutes**

## MODIFIED PETROFF’S METHOD

**PROCEDURE**
**To x ml of sputum add 2x  ml of 4% NaOH**
**Tighten cap of container and shake to digest**
**Let stand for 15 minutes at RT with occasional shaking**
**Centrifuge at 3000 g for 15 minutes**
**Carefully pour off the supernatant**
**Add approx. 20 ml sterile distilled water  and re-suspend the sediment**
**Centrifuge again at 3000 g for 15 minutes **
**Decant supernatant and inoculate deposit on to two slopes of L-J  and one slope of L-J with pyruvate**

## PETROFF’S METHOD

- Advantages:
**Simple, inexpensive & control the growth of contaminants**
**Twenty samples can be processed in 2 Hrs, with centrifuge capacity being the limiting factor**
**Sterilized NaOH can be kept for several weeks**
** **
- Limitations:
**The specimen exposure times must be strictly followed to prevent over kill of tubercle bacilli. The initial kill is independent of additional contributory factors such as heat build-up in the centrifuge and centrifugal efficiency**

## Processing of sputum with CPC Method

**If delay of more than 48 hours between collection and processing is anticipated, the sputum should be collected with 1%CPC and 2%NaCl2**
**CPC acts as homogenizing and decontaminating agent **
**It helps in retaining viability of Tubercle bacilli up to 7 days**
**These specimens should not be treated with NaOH ( Petroff’s)**

## Processing of CPC containing samples

**To the specimen with CPC, add 15-20 ml sterile distilled water (to reduce the viscosity) **
**Tighten cap of container and mix well by inversion**
**Centrifuge at 3000 g for 15 minutes**
**Carefully pour off the supernatant**
**Add approximately 20 ml sterile distilled  water and re -suspend the sediment**
**Centrifuge again at 3000 g for 15 minutes **
**Decant supernatant and inoculate deposit on to two slopes of L-J medium and one slope of L-J with pyruvate**

## Egg based Medium

**Advantages**
**Easy to make and inexpensive**
**Supports good growth of tubercle bacilli**
**Can be stored in refrigerator for several weeks**
**Other Factors:**
**Fresh eggs to be used**
**Bottle caps to be to tightly closed to minimize drying**
**Inspissation and Malachite green suppresses the growth of Non Mycobacterial organisms**
**Disadvantages**
**Prolonged delay   8 weeks!**
**Contamination covers usually entire surface - recovery difficult**

## Colony Morphology of M.tuberculosis

- Dry wrinkled warty     growth.
- Eugonic

## Reading and Reporting

**Characteristics of Tubercle bacilli**
**Growth of Primary culture takes 2 – 4 weeks to obtain visible colonies**
**Colonies are buff colored and rough, having the appearance of bread crumbs or cauliflower**
**Not easily emulsified but give a granular suspension**
**Microscopically frequently arranged in serpentine cords of varying length or show linear clumping**

## Reporting of culture results

**	****Reading**
**No Growth**
**1 – 19 colonies**
**20-100 colonies**
**>100 colonies**
**Confluent growth**
**Contaminated**

**Report**
**  ****Negative**
**  ****Positive         ( No.of colonies)**
**  ****Positive           (1+)**
**  ****Positive           (2+)**
**  ****Positive            (3+)**
**  ****Contaminated**

## Culture: Extra-Pulmonary Samples

**Aseptically collected samples**
- Body fluids:
- Spinal ,Pleural, Pericardial, Syanovial, ascitic, Blood, Pus & Bone marrow
- Tissues:
- Lymph node, Needle biopsies or  Tissue biopsies
- Specimens known to contain contaminating flora:
- Gastric lavage, Bronchial washings & Urine

## Extra-Pulmonary samples

** ****Pauci-bacillary**
**	****	****	****Direct inoculation**
**	****	****	****Milder decontamination **
**	****	****	****5% H2SO****4****treatment for most   specimens**
** ****Heavily contaminated****  **
**         ****Pus,   Gasric aspirate - Petroff’s method**
**Media:**
**	****	****	****LJ, LJP & SKLM  (PACT or PANTA )**

## Culture

**Conventional****:**
** ****LJ**
** ****LJP**
** ****7H10**
** ****7H11**
** ****7H9 **
** ****Kirchner’s****      **

## Other Culture Methods

**Septi-check AFB****  :    Non radiometric, Non ****	****	****	****automated**
**MGIT 960****              :      Automated.  Monitors ****	****	****continuously every 60min. O****2**** utilization, ****	****Intensification of O****2**** quenching fluorescent dye**
**MB/Bact****                :      Non radiometric, ****	****colorimetric detection of CO****2**
**ESP Culture ii****       :      Reliable non radiometric ****	****alternative system**
**M****icroscopic ****O****bservation of ****B****roth ****C****ulture & ****MODS**
**M****icro ****C****olony ****D****etection ****S****ystem**

## BACTEC 460 TB System

**Developed in 1969 by Deland and Wagner.**
**Principle**
**When the BACTEC 12B vial is inoculated, mycobacteria, if present, utilize the ****14****C labelled substrate (fatty acid) and release ****14****CO****2****. **
**The BACTEC instrument measures quantitatively the radioactivity on a scale ranging from 0-999, as  GI. **
**The daily increase in GI is directly proportional to the rate and amount of growth in the medium. **
**When an inhibitory agent is introduced in the medium, inhibition of metabolism is indicated by reduced production of ****14****CO****2 **** and decrease in GI. **
**This basic principle is utilized**** ****in drug susceptibility and identification tests in this method. **

## Recovery rate (%) of Mycobacteria from positive specimens by different systems

| Mycobact. species | MB/BacT | B 460 | LJ |
| --- | --- | --- | --- |
| MTB 148 | 125 (84.5) | 135 (91.2) | 105 (70.9) |
| NTM (31) | 16 (51.6) | 21 (67.7) | 10  (47.1) |
| Total 179 | 141 (78.8) | 156 (87.1) | 115 (64.2) |
| All clin.sig.
Myco(162) | 135 (83.3) | 144 (88.9) | 113 (69.7) |

**Recovery rate (%) of Mycobacteria from positive specimens by different systems**

*Piercimoni C. et al. J.clin.microbiol.2001,*_*89*_*,651-659*

## Various culture methods for recovery of Mycobacteria from clinical samples

| organisms | No. (%) Isolates detected |  |  |
| --- | --- | --- | --- |
|  | MGIT/LJ | MB/BacT+LJ | B 460 +LJ |
| ALL (120) | 106(88.3) | 106 (88.3) | 103 (85.8) |
| M.TB complex (96) |   88 (91.7) |   88 (91.7) |   83 (86.5) |
| All NTM sp.(24) |   18 (75.0) |   18 (75.0) |   20 (83.3) |
| M.kansasii 
genotype I (14) |   12 (85.7) |   14 (100.0) |   14 (100.0) |
| M.xenopi (5) |     4 (80) |     2 (40.0) |     3 (60.0) |
| Detection time(days) | 12.6 | 15.9 | 11.8 |

*Alcaide F  J.Clin. Microbiol. 2000.  398-401 Vol38.No.1*

## MODS versus  other culture methods*

| 
Method | Pos.each
Method(%) | Pos. by
atleast one cult.(%) | Sens.   % | Median
detection
days |
| --- | --- | --- | --- | --- |
| Auramine 0 | 76 | 98 | 78 |  |
| MODS | 89 | 97 | 92 | 9 (4-31) |
| MGIT | 88 | 95 | 93 | 10 (3-39) |
| LJ | 73 | 96 | 76 | 24 ( 6-59) |
| Micro COL 7H11 | 75 | 96 | 78 | 14.5(4-28) |
| PCR | 81 | 90 | 90 |  |

**** Based on 172 samples***

*Caviedes.L. et al   J..Clin.Microbiol. 2000, *_*38*_*, 1203*

## Cost: MODS versus other methods

| Method | Detection cost ($) | Susceptibility cost ($) |  |
| --- | --- | --- | --- |
|  |  | Two drugs | Four drugs |
| MODS | 0.77 | 1.72 | 1.80 |
| MGIT | 7.00 | 35.02 | 63.03 |
| BACTEC | 2.55 | 12.75 | 23.00 |
| LJ | 0.14 | 1.60 | 1.57 |
| Micro 7H11 | 0.29 | 1.60 | 2.92 |
| MABA | 1.23 - 2.43 | 5.62 | 6.87 |
| PCR | 2.90 | - | - |
| AFB smear | 0.10 |  |  |

*Caviedes.L. et al.  J.Clin. Microbiol. 2000, *_*38*_*, 1203*

## Identification of M.tuberculosis

-  **No single  test can differentiate *****M.tb *****from other mycobacteria.  **
**The following tests,  along with the colony morphology may  enable an identification >95% of the *****M.tb***** strains:**
**Susceptibility to p-nitrobenzoic acid (PNB)**
**Niacin production test**
**Catalase activity at 68****o****C/pH 7**
**Nitrate reduction test (optional)**

## Susceptibility to p-nitrobenzoic acid (PNB)

**Procedure:**
- Weigh out 0.5g PNB and dissolve in the minimum amount of dimethyl formamide  (~ 15ml).
-  Add to 1 litre of L-J fluid, distribute in 6 ml amounts and inspissate. Store in the cold.
- Inoculate with the neat bacterial suspension two slopes of LJ medium without drugs and one slope of LJ medium containing PNB at a conc. of 500mg/ l and incubate at 370C. Read after 28 days.
**Results and interpretation:**
*M.tb* does not grow on PNB medium.  All other mycobacteria are resistant.

## NIACIN PRODUCTION TEST

- Although all mycobacteria produce niacin, comparative studies  show M.tb accumulates the largest amount of nicotinic acid and its detection is useful for  diagnosis.
-  Niacin negative M.tb strains are rare, while very few other mycobacterial species yield positive niacin tests.
- Cultures grown on LJ with asparagine yield consistent results in the niacin test and hence LJ is preferred.
-  A culture must be at least three to four weeks old and must have sufficient growth of at least 100 colonies.

## NIACIN PRODUCTION TEST

- Reagents
- o-tolidine, 1.5%
-               o-tolidine                  1.5 g
-               Ethanol                   100ml
- 		Mix in an amber bottle and store in the dark in the refrigerator, prepare fresh weekly.
- Cyanogen bromide solution, approx. 10%.
- 		A saturated aqueous solution of cyanogen bromide is approx 10%. Store at 4oC in the refrigerator.

## NIACIN PRODUCTION TEST

**Procedure:**
**Check for water of condensation in the culture tube. If needed add 1 ml sterile distilled water to the tube.**
**Place the bottles horizontally in the autoclave at 121****o****C**
**Allow 30 minutes for the extraction of niacin**
**Place the bottles upright for 5 minutes to allow the fluid to drain to the bottom**
**Remove 0.25 ml of the extract to a clean screw-capped tube**
**Sequentially add 0.25 ml of o-tolidine and 0.25 ml of 10% cyanogens bromide. Mix well**
**Close the tube and observe the solution for the formation of a pink color (positive) within 5 minutes**
**Add 2-3 ml of 4% NaOH to each tube and discard.**

## CATALASE TEST at 68oC/pH 7.0

**Catalase is an intracellular, soluble enzyme capable of splitting hydrogen peroxide into water and O****2****.**
** ****Mycobacteria posses several kinds of catalase that vary in heat stability.**
**The O****2**** bubbles into the reaction mixture to indicate catalase activity.  **
** ****All mycobacteria possess catalase enzymes, except for certain INH res. mutants of M.tb and M.bovis**
** ****Quantitative differences in catalase activity can be demonstrated by 68****0****C test at pH 7 (indicates loss of catalase activity due to heat). **
**Drug susceptible strains of M.tb lose catalase activity when heated to 68****0****C for 20 minutes. **
** ****For these tests cultures on LJ should be used. **

## CATALASE TEST at 68oC/pH 7.0

**Reagents**
**Phosphate buffer solution, 0.067M (M/15)pH 7.0**
**	****	****Dissolve 9.47 g disodium phosphate in 1 litre water to provide 0.067 M solution (Solution 1)**
**	****	****Dissolve 9.07 g potasium dihidrogen phosphate  in 1 litre water to give 0.067 M KH****2****PO4 solution    (Solution 2)**
**Mix 61.1 ml of solution 1 with 38.9 ml of solution 2. Check pH.**
**Hydrogen peroxide 30% solution****. Store in the refrigerator. **
**Tween-80, 10%****        Mix Tween-80 with 90ml distilled water and autoclave at 121oC for 10 minutes. Allow to cool. Store in the refrigerator.**
**Complete catalase reagent (Tween-peroxide mixture):**
**	****Immediately before use, mix equal parts of 10% Tween-80 and 30% hydrogen peroxide.  0.5 ml reagent for each  test.**

## CATALASE TEST at 68oC/pH 7.0

**Procedure**
**With a sterile pipette, aseptically add 0.5 ml of 0.067 M buffer into 16 x 125 mm screw-capped test tubes**
**Suspend a loopful of the test culture in the buffer solution, using a sterile loop**
**Place the tubes with the emulsified culture in a previously heated water bath at 68****o****C for 20 minutes. Time and temperature are critical**
**Remove the tubes from heat and cool to RT. Add 0.5 ml of freshly prepared Tween-peroxide mixture to each tube**
**Observe the formation of bubbles appearing on the surface of the liquid. Do not shake the tubes as it will  result in false positives.**
**Hold negative tubes for 20 minutes before discarding **

## NITRATE REDUCTION TEST

**Reagents**
** ****(1) Substrate: 0.1M sodium nitrate in buffer**
**                 ****Sodium nitrate                      0.85 g**
**          ****M/45 phosphate buffer, pH 7.0         100 ml**
**	****	****Prepare M/45 buffer by diluting M/15 buffer 1:2 with d. water. Dissolve the sodium nitrate in buffer and distribute in 2-ml amounts in screw-capped test tubes. Sterilize at 15 lbs pressure for 15 min. and store in the cold.**
**(2) Hydrochloric acid (50 %) in d. water**
**(3) Sulphanilamide, 0.2% : 0.2 g in 100 ml d. water**
**(4) Coupling agent **
**            ****N- (1-naphthyl)-ethylene diamine di-HCl     0.1 g**
**            ****Distilled water                                              100 ml**
**(5) Zinc dust**

## NITRATE REDUCTION TEST

**Procedure**
- Emulsify a loopful of growth from an actively growing culture in one tube of the substrate Incubate in a water bath at 37oC for 2 hr. Remove from the water bath and add in the following order:
-      	 I drop of 1:1 HCl
- 	2 drops of sulphanilamide solution
-         2 drops of the coupling agent
- Development of a pink to deep red color indicates a positive reaction. If no color develops, report as negative.

## NITRATE REDUCTION TEST

- Confirm all negative reaction by adding a pinch of zinc dust. Development of a pink color at this stage indicates that the initial negative reaction was genuine. If no color change occurs after adding the zinc dust, the reaction has proceeded beyond nitrite into other components. Repeat the entire test.
**Controls**
- Use strain *M.tuberculosis*, H37Rv as positive control and a reagent control without organisms as negative control.

## Identification of M. tuberculosis

**SUMMARY**
- Growth temperature 35o-37oC only
- No pigmentation
- Niacin positive
- Catalase negative at 68oC
- No growth on LJ medium containing PNB
- Positive reaction for nitrate reduction

## Differentiation of Mycobacteria

|  | M.tuberculosis | NTM |
| --- | --- | --- |
| Colony morphology | Rough, eugonic | Mostly smooth |
| Growth at 37oC | + | +/ - |
| Growth at 25oC | - | + |
| Pigmentation | - | +/ - |
| Niacin | + | - |
| PNB | - | + |
| Nitrate reduction | + | - |
| Catalase at 68oC | - | + |