# Presentation: 474356

## Slide 1

**Notes:**
“An Ancient disease is killing more people today
than ever before. Tuberculosis, which many of us
believed would disappear in our lifetime, has staged
a frightening comeback.
We are at the crossroads.
We can permit the TB epidemic to become more
deadly, or we can act now to end the suffering and
deaths.
We are at the crossroads.”
G. Harlem Brundtland, M.D., Director, WHO 1998
As we enter the twenty first century, many diseases
continue to challenge the health of people around the
world. Tuberculosis, both an ancient and a modern
disease, looms in the foreground.

Over 8 million cases of TB continue to occur annually.
Close to 2 million people still die of TB each year.

Poverty and malnutrition fuel the fire of disease in
developing countries, while migrations, wars, and natural
catastrophes set the scene for mass infection.

The global escalation in TB,the rise in HIV and the
emergence of multiple drug resistant strains of TB have
caused urgency and concern.

Several international agencies are dedicated to the control and eradication of tuberculosis worldwide.

Practical and affordable diagnosis is a key link in the chain.
ACID-FAST DIRECT SMEAR MICROSCOPY

This teaching module will focus on the Ziehl Neelsen
diagnostic technique of direct smear microscopy of sputum.

Since most TB cases are pulmonary, sputum smear
microscopy is the most available tool for TB diagnosis.

It is inexpensive, rapid and simple to perform.

More importantly, it can detect the most infectious cases of
tuberculosis.

Identifying these cases can lead to treatment and cure while
interrupting the flow of TB transmission. This procedure can
also assess response to drug therapy and monitor cure rates
after treatment.\
There are many facets to the process of smear microscopy.

First let’s discuss Biosafety.
Working with TB requires care and special precautions.

A brief review of the mode of transmission will highlight the
dangers in working with this microbe.

## Slide 2

**Notes:**
Mycobacterium tuberculosis
...is spread through the air from person to person via
coughing, sneezing, and even singing and speaking.

Tiny invisible droplets dry quickly to form droplet nuclei that
are less than 5 microns in diameter. They can contain disease
organisms such as tubercle bacilli. These droplets may remain
suspended in the atmosphere for several hours. Another
person can breathe in the particles and become infected.

This is especially dangerous in a confined area. Keep in mind,
when appropriate techniques are employed, there is very little
risk of acquiring infection in the laboratory.

It is vital that all personnel be aware of potential hazards and
work in a careful manner. 

## Slide 3

**Notes:**
The greatest risk of infection in the laboratory involves sputum
collection. Preparing smears presents less risk to the lab
worker than exposure to direct coughing.

Since TB patients are sometimes directed to the lab for sputum collection, precautions must be taken to minimize the risk of exposure.

Never collect sputum specimens inside the
clinic or laboratory.

## Slide 4

**Notes:**
 It is safer to collect the specimens outside.

At all times, avoid creating aerosols in the lab!
A daily routine that is followed exactly will help
maintain safe conditions.

Strict adherence to safety regulations in the laboratory is
crucial. Safe practices include:

        Washing hands frequently and always before and after
      performing any procedures.
        Establishing airflow in working areas that will carry
      infectious particles away from lab personnel. This air
       should be exhausted into a remote location.
        Strictly refraining from smoking and eating in the lab!!!
        Wearing protective clothing and use of protective
      equipment.

## Safety

- Washing hands frequently
- Airflow
- No smoking or eating in lab
- Wearing protective clothing and
-    use of protective equipment.

## Slide 6

**Notes:**
Surgical masks DO NOT protect against TB infection !

Effective respiratory protection, such as an N95 respirator, is
expensive, and probably unnecessary if the technician uses
appropriate technique.

Careful handling during smear preparation should produce
minimal aerosols.

The low risk of infections during these procedures may not
justify the cost of respirators and gloves where there may be
limited resources for TB control.

Each country must evaluate the risks and decide on the level
of protection that is appropriate with the resources that may
be available.
In this training video, the technicians do not wear gloves or
masks.  Hand-washing and careful technique are acceptable
practices for most countries. 
Remember, safety is everyone’s concern
no matter what job is being performed.

## Slide 7

**Notes:**
Laboratory Arrangement

To perform TB microscopy, a laboratory must have three areas:

## Slide 8

**Notes:**
Area 1
 a well lighted space with a sink or basin with running water
for preparation and staining of smears,

## Slide 9

**Notes:**
Area 2
a microscope table, (if there is no electricity, this table should
be placed directly in front of a window) 

## Slide 10

**Notes:**
Area 3
and a table for recording results and slide storage. 

## Slide 11

**Notes:**
Specimen Collection
Two kinds of containers are recommended for
sputum collection:

## Slide 12

**Notes:**
The first is a disposable wide-mouthed, screw-capped
container. It is made of unbreakable transparent plastic. It
must be hermetically sealed to keep the specimen from leaking
and drying out.

The second is a screw capped “Universal container”
made of heavy glass which can be first sterilized, cleaned
and used again.

## Slide 13

**Notes:**
Many countries use their own containers. 

## Slide 14

**Notes:**
Labeling of Specimens

It is critical to label a specimen properly to avoid confusion
and delay.

A request form must accompany each specimen. The
information on the form must exactly match the information
on the container. Always label the container on the side, never
on the lid.

Using an indelible marking pen, write the name of the TB
suspect and the date of collection. 

## Slide 15

**Notes:**
Sputum Specimens

For best recovery of tubercle bacilli, collection
of three specimens is advised   At least one should
be an “early morning” specimen.

The patient should be properly instructed to ensure that the
collected specimen is of sputum, not saliva.

Clear saliva or nasal discharge is not considered a suitable
specimen for TB. Nevertheless they should be processed.
Specific instructions for collection can be found in the TB
program manual.

A good sputum specimen should be 3-5ml in quantity. It is
frequently thick and mucoid, but may be fluid with chunks of
dead tissue. The color may range from opaque white to green.
Bloody specimens will be reddish or brown in color.

Record the visual appearance of the sputum sample on the
lab form.

## Slide 16

**Notes:**
Washing Hands

Always wash hands with soap and water after
handling specimens and containers. 

## Slide 17

**Notes:**
Transport of Specimen

Specimens should be packed and transported according to
your country’s NTP guidelines.

Specimen Handling

Open the transport box carefully and examine it for cracked
or broken containers. Discard any broken or leaking container
and request another specimen.

All specimens must correspond with the accompanying list
containing data of the TB suspect.

Once verified, record all information in the laboratory
register.

## Slide 18

**Notes:**
The Laboratory Register

The WHO or IUATLD laboratory register is strongly recommended as a guideline. The format of the register should
never be altered.

## Specimen Information

- Laboratory serial number
- Date received
- Patient’s name, sex, age, & address
- Name of Health Institution
- Reason for exam

**Notes:**
Information for each specimen must be complete, precise,
and include:

Laboratory serial number
Date received
Patient’s name, sex, age & address
Name of Health Institution
Reason for exam (whether as diagnosis or as
    chemotherapy follow up).

Label the specimen container accordingly. 

## Slide 20

**Notes:**
Smear Preparation

To ensure a safe and consistent workflow, always arrange
equipment and materials in the same way at the workbench.

## Slide 21

**Notes:**
The following supplies are required:
Forceps,
A diamond marker or lead pencil,
Wooden applicator sticks or loop,
A sand/alcohol jar (if a loop is used),
A discard container with disinfectant,
A Bunsen burner or spirit lamp,
A box of new slides, and
Specimen containers. 

## Slide 22

**Notes:**
Always use new slides when preparing AFB smears.
They should be cleaned with alcohol and wiped dry...
or passed briskly through a flame.

This will remove any residue of oil that could interfere with
staining.

Never reuse sputum smear slides for TB work!!! 

## Slide 23

**Notes:**
Properly identify each slide

Properly identify each slide with the laboratory and specimen
number. Use a lead pencil to write these numbers on the frosted end of the slide. 

## Slide 24

**Notes:**
If the slide is not frosted, engrave the laboratory serial number
using a diamond pointed stylus.

Never use a grease pencil, since markings could wash off
during the staining process.

## Slide 25

**Notes:**
Open the container slowly to avoid producing aerosols.

Using either a wooden applicator stick or a loop, pick out
portions of sputum for examination. Solid caseous particles
often produce the highest number of bacilli.

## Slide 26

**Notes:**
Using a continuous rotation, cover an oval area approximately
2 cm in length on the slide. Put only one smear on each slide. 

## Slide 27

**Notes:**
How can you tell you have the proper thickness?

If it is too thin, the specimen may yield false negative results.
If it is too thick, the smear may wash off the slide during the
staining procedure. 
Newsprint can usually be read behind a
dried smear when it is the proper thickness.
If a wire loop is used, clean it before the next use. Dip the
contaminated loop into a sand and alcohol bottle and move it
up and down to remove solid debris.

Next, heat the wire loop in the flame until red hot. Allow it to
cool before using it again.

Wet slides can create aerosols if disturbed. Place them in a
protected area where they can air dry for 15-30 minutes.

Do NOT flame them to induce drying.
This can also produce aerosols.

## Slide 28

**Notes:**

When dried, fix the slides using the blue flame of a Bunsen
burner. With forceps, pass the slide briefly through the flame
3 times, smear side up.

Heat fixation ensures that the sputum will stick to the glass
slide.  Excessive heating could damage the bacilli. If not
sufficiently heat fixed, the acid fast bacilli may wash off during
staining.

## Slide 29

## Slide 30

**Notes:**
Staining Procedure

Since tubercle bacilli retain the primary stain even after
exposure to strong acid solutions, they are called acid-fast.
In the Ziehl Neelsen staining procedure, using carbol fuschsin
and methylene blue, the acid-fast organisms appear red.

## Slide 31

**Notes:**
Prepare the area for staining.

On the table counter next to the sink, place the following items:
Staining reagents, clock, forceps, cotton/alcohol holder or
Bunsen burner, and tray containing fixed slides to be stained.
A slide rack should be placed in the sink.

## Standard reagents:

- Carbol Fuchsin
-  Acid/Alcohol or Sulfuric Acid
- Methylene Blue

**Notes:**
To assure quality and consistency, many countries produce
reagents in a central lab. If preparation instructions are
required, consult the WHO and IUATLD manual for standard
guidelines.

Standard reagents are:
        carbol fuchsin
        a decolorizing solution of acid/alcohol or sulfuric acid
        the counterstain methylene blue.

All reagents must have expiration dates.
Discard all expired dates.

## Slide 33

**Notes:**
Filter carbol fuchsin prior to use. If precipitate is
still detected, discard the stain.

## Slide 34

**Notes:**
The staining procedure should be posted above
the staining area.

To avoid cross contamination, never stain AFB smears in a
staining dish.

Now, let’s study the important steps of the staining procedure. 

## Slide 35

**Notes:**
Using forceps, place slides on a staining rack with the smear
side up. Place all slides in uniform orientation.

Slides must not touch each other. This avoids the possibility
of stain running over and causing cross-contamination. Include
a + and - control slide daily for quality control purposes. Never stain more than 12 slides at a time.

## Slide 36

**Notes:**
Cover the entire surface of each slide with carbol fuchsin.
Using a Bunsen burner or cotton and alcohol flame, gently
heat the slides until vapor rises.

Do not allow them to boil or dry. Boiling will
alter the shape of the TB bacilli and could result
in a false negative reading.

Allow the stain to remain on the slides for 5 minutes. Maintain
heat throughout this period. Adequate time is required for
the carbol fuchsin to penetrate and stain the cell wall.

## Slide 37

**Notes:**
Gently wash the stain from each slide with a stream of cold
water until all free stain has washed away. Always rinse gently
so the smear is not washed off of the slide.
Individually tilt each slide to drain off any excess rinse water.
This prevents puddles remaining on the slide which can dilute
the next reagent.

## Slide 38

**Notes:**
Cover each slide with decolorizing solution such as acid
alcohol.

Leave this on the slides for 3 minutes. If under-decolorized,
sputum contents other than the TB bacilli may remain stained.
This could lead to a false positive result.

## Slide 39

**Notes:**
Rinse slides again carefully with water and tilt each slide to
remove excess water.

If the slide is still pink, an additional amount of decolorizing
solution can be reapplied for 1 to 3 minutes.

## Slide 40

**Notes:**
Next, counter-stain with methylene blue for l minute 

## Slide 41

**Notes:**
Rinse slides again carefully with water and tilt each slide to
remove excess water.
Finally, tilt each slide and place in a slide block to air dry.
Do NOT blot.

After smears are stained, clean off the back of each slide if
necessary with some alcohol on a paper towel.

Do not examine slides until they are thoroughly dried.

Cover the stained slides to protect them from sunlight which
can fade acid-fast bacilli.

## Slide 42

## Slide 43

**Notes:**
Microscopic Examination

A good quality binocular microscope is
recommended for reading TB smears.

It must be equipped with an electrical light 
source or mirror, an oil immersion or 100X objective, eyepieces of 8-10X magnification and a mechanical stage.

Daily care and cleaning are essential to avoid dust, oil build
up and moisture.

An area of subdued lighting is preferable for reading slides.

However, if no electricity is available, daylight must be used
as a light source and the microscope should be placed in front
of a window.

## Slide 44

**Notes:**
Arrange the following supplies next to the microscope: a
bottle of immersion oil, organic solvent, lens paper, laboratory
register and a slide box for storage.	

Use the 40X objective to focus and determine a suitable
reading area of the slide.

Place a drop of immersion oil on the stained
smear. Let the drop fall freely onto the slide.

Never touch the slide with the oil applicator. This
could lead to carry over of AFB in the immersion oil to
the next slide.

Turn the nosepiece to bring the 100X objective into place.
Now, gently lower the 100X objective.

It should barely touch the oil. Never allow the lens to touch
the slide. This can damage the lens and possibly break the
slide.
While looking through the eye-piece, adjust the immersion
lens slowly and focus until the image on the smear appears.
To fine focus, turn the fine adjustment knob carefully.

## 100 fields!!

Examining fewer than 100 fields can cause a false negative report.

- Examining fewer than 100 fields can cause a false negative report.

**Notes:**
At least 100 microscopic fields should be examined before reporting as a negative smear. Examining fewer than 100 fields may cause a false negative report. Fewer than 100 fields can be read if the slide is positive for AFB.

Read systematically to avoid overlap, moving the slide
lengthwise so that the next field to the right can then be
examined.

## Slide 46

**Notes:**
Tubercle bacilli resemble fine red rods standing out against
the blue background. They may be slightly curved, granular,
and may occur singly, in pairs, or groups.
Count the number of AFB per field and record this figure on
the lab report.

After examining the slide thoroughly, remove it carefully from
the microscope stage.

Check the slide number again and enter the result immediately
into the lab register.

## Slide 47

**Notes:**
After completing the reading, remove the oil from the slide
with organic solvent.

When dried, place the slides carefully in a slide storage
container for transfer to a referral laboratory for quality
control. This must be done on a regular basis as determined
by the National Tuberculosis Programme.

## Slide 48

**Notes:**
Before examining the next slide, wipe the immersion lens
with lens paper. This will protect from carry over of organisms
that could create a false positive reading.

Handle specimens carefully!

All sputum specimens are considered potentially infectious.
Save all specimens until the smears have been examined and
recorded; then sterilize and discard them. Disposable
containers must be used only once.

## Discard of Specimens

- Burning
- Boiling
- Autoclaving

## Slide 50

**Notes:**
Recording and Reporting

It is vital that results of all sputum exams
performed be entered correctly into the
Tuberculosis Laboratory register. Enter positive
results in red, for quick reference.

If acid-fast bacilli are seen, report the observation as “smear
positive for AFB”. These patients are infectious and must be
registered for treatment with an effective regimen.

Testing and reporting should be done as soon as possible,
preferably within 24 hours.

## WHO and IUATLD

- Negative Report:  “Negative for
-    acid-fast  bacilli where no organisms have
-    been observed in 100 fields.
- Positive Report:  “Positive for acid-fast bacilli :  Provide AFB quantitation.

**Notes:**
The WHO and IUATLD recommended method for reporting
results is as follows:

Negative: Report: “Negative for acid-fast
bacilli” where no organisms have been observed
in 100 fields.

Positive: Report: “Positive for acid-fast bacilli.”
Provide AFB quantitation.

The number of AFB found is an indication of the degree of
infectivity as well as severity of disease. Results must be
quantitated.

## Semi-quantitative reportingZiehl Neelsen

-  No AFB are found in 100 fields:
-    “No acid-fast bacilli observed.”
-  1-9 /100 fields:         Report the exact figure.
-  10-99 AFB /100 fields, 		    1+
-  1-10AFB /field, 			    2+
-  Greater than 10 AFB/ field, 	    3+

**Notes:**
The following semi-quantitative reporting method is
recommended for the Ziehl Neelsen technique.

If No AFB are found in 100 fields, report:
No acid-fast bacilli observed.
If 1-9 are found in 100 fields:
record the exact figure.
If 10-99 AFB are found in 100 fields:
report as 1+.
If 1-10AFB are found per field, report as 2+.
f greater than 10 AFB per field, report as 3+.

## Information

- Evaluation of the quality of the specimen
- Staining method used (Ziehl Neelsen)
- Smear result
- Date of exam
- Signature of microscopist

**Notes:**
Include the following information in the report:

Evaluation of the quality of the specimen
Staining method used (Ziehl Neelsen)
Smear result
Date of exam
Signature of microscopist

There are many species of AFB in addition to the tubercle
bacilli. Do not try to identify species by microscopy. Report
only the number of AFB seen.

After completing the lab report, send it back to the health
care center for further action.

## Slide 54

## Slide 55

**Notes:**
Quality Control in the TB Laboratory

Quality Control (QC) should be routinely
performed in the laboratory to ensure
reliability as well as reproducibility of results.

Perform QC on each new batch of stain. Observe these results before reading patient slides. This important step will verify correct staining procedure and microscope function.

When controls are not demonstrating proper staining,
determine the cause and correct it.

If the negative control appears red after the staining procedure, this is the result of incomplete decolorization. 

## Slide 56

**Notes:**
If the positive control slide does not show AFB, either the
staining reagents are defective or the procedure has been
performed incorrectly. When the problem has been identified
and corrected, repeat the staining procedure with new slides
and controls.

Each phase of smear microscopy must be carefully performed.
Errors made during any step can lead to incorrect results.

## Some reasons for false positive results:

- Errors in specimen handling or recording information
- Re-use of containers or positive slides
- Unfiltered fuchsin
- Contaminated immersion oil
- Inadequate decolorization

**Notes:**
Some reasons for false positive results include:

Errors in specimen handling or recording
information
Re-use of containers or positive slides
Unfiltered fuchsin
Contaminated immersion oil
Inadequate decolorization

## Some reasons for false negative results:

- Errors in specimen handling or recording information
- Poor quality sputum
- Excessive decolorization
- Reading less than 100 microscopic fields

**Notes:**
Some reasons for false negative results include:

Errors in specimen handling or recording
information
Poor quality sputum
Excessive decolorization
Reading less than 100 microscopic fields

## Slide 59

**Notes:**
AN ERRONEOUS REPORT CAN HAVE
DEVASTATING RESULTS!

If a False positive result is reported, a patient will be placed
on treatment unnecessarily.

If it is a Follow-up, treatment is lengthened. Valuable
medication is wasted and, sadly, it can cause emotional trauma
to patients and their families. Patients may lose confidence in
the program itself.

If, on the other hand, a False negative result is reported, a
catastrophic set of events can occur.
        A patient with TB is not treated.
        If the sample was a follow-up specimen, the intensive
    phase of therapy would not be extended causing
      inadequate treatment. This can lead to additional
      suffering, spread of TB to family and community, and
      even end in patient death.

Accuracy at every stage is vital to the health and well being
of many people.

As a member of the global campaign to
control this dreaded disease, the role of the
laboratory technician is as significant as every
other member of the TB control team.

If the important control and prevention
programs are to be successful worldwide,
accurate smear microscopy is a critical link in the
chain of tuberculosis control.

We are at the Crossroads.