STUDENT PERSONAL REPORT

PRACTICAL SKILL

Ziehl Neelsen Staining

Name BHELVAA ZAHIRA ZAHRA

NIM 225070107111003

Class (A/B/C) A

Group (1-24) 5

BASIC MEDICAL SCIENCE 4 A

FACULTY OF MEDICINE UNIVERSITAS BRAWIJAYA

2023
General Instruction:

1. This report is a personal report. You can work on the report together with your friend but
each student needs to submit the report into the designated Google Classroom.
2. You have 72 hours to finish this report and submit it to Google Classroom
3. This report is your ticket to enter BMS4A OSCE at the end of even semester
4. During this skill session, work carefully and keep in mind that you are facing a highly
infectious bacteria
5. Wash your hands and clean your gadget properly after this skill session
Literature Review:

Acid-fast bacilli has waxy material on the cell wall called mycolic acid that makes it difficult to
stain but also difficult to de-stain once it is stained. In this skill session we use the method of
ZIEHL NEELSEN.

Below is the principle of acid fast staining:

Figure 1. Principle of Ziehl Neelsen staining

In summary, in order to disrupt the wax, you need to add two intensifiers, chemical and physical
intensifiers. These two additions will enable the stain to get into contact with the cell wall and
stain it. Once the wax cools down, it will be back to its original shape surrounding the cells, and
this will trap the fuchsin stain inside the wax. The stain will remain even when the cell comes in
contact with acid alcohol (alcohol 96% + H2O2 3%) as the decolorizer agent. Other cells who do
not have the wax will release the stain at decolorizing period, and will be stained by counterstain
(methylene blue).
The sequence of Ziehl Neelsen acid fast staining is as follow:

Figure 2. Step by step for Ziehl Neelsen

Figure 3. Result of Ziehl Neelsen staining

The expected result of this stain should be the appearance of red rods with a blue background.
The blue background is caused by inability of the non-waxy cells to retain fuchsin during primary
staining. When you need to count the quantity, Count the number of acid fast bacilli under the
microscope using IUAT / WHO scoring. Furthermore, to ensure diagnosis of TBC, one should
consider the culture of Mycobacterium tuberculosis. There are several things to consider about
this particular culture:
• Optimum growth temperature at 35 – 370C.

• The specimen is incubated until 8 weeks at most, and growth is reported each two weeks. If
there is no growth until 8 weeks, then the person is declared not infect
• For rapid growers, the growth can be less than 7 days.

• Growth is better 5 – 10% CO2

Two of the most used culture medium for this bacteria is Lowenstein Jensen (LJ) and
Middlebrook medium. Lowenstein Jensen consists of malachite green, glycerol, asparagine,
starch, coagulated egg, and mineral salts. Middlebrook consists of oleic acid and albumin as
key ingredients, which protect Mycobacterium from toxic agents, helping for the growth of
tubercle bacilli. When grown on LJ medium, M. tuberculosis appears as brown, granular
colonies (sometimes called "buff, rough and tough"). The example of culture results on LJ
medium are shown in Figure 4. The example of culture results on Middlebrook medium are
shown in Figure 5.

Figure 4. LJ culture result

Figure 5. Middlebrook culture result

STUDENT PERSONAL REPORT
P2 BTA ENDOSPORE ANAEROB

CASE
A 45 yo man came to the outpatient department of your hospital complaining of an unresolved
cough which started three months ago, accompanied by significant weight loss, also night
sweating and shivering. You decided to examine her sputum because you could not rule out the
possibility of tuberculosis.

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Note:
Questions will be written with red fonts. Fields that need answering are marked by “………”
Please answer using black fonts.
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Question Chain 1
Explain as clear as you can about how to do Ziehl Neelsen staining (type your answer
into the following box)
Please mention all the important things you need to take into account when doing the
procedure!
(Can be finished before practical work)
Tools and Materials:
 Carbol Fuchsin
 Water
 Alcohol Acid
 Methylene Blue
 Bunsen Burner
 Spiritus
 Slide
 Mycobaterium tuberculosis colonies
 Lighter
 Towel / Tissue
 Stopwatch
 Inoculating loop

Steps :
 Prepare the slide, heat the slide with heating and after that spread the colonies of the
acid-fast bacteria using inoculating loop. Make sure that the inoculating loop also
going through the process of heating.
 On top of the slide, inundate the colonies on the slide with carbol fuchsin.
 Heat the slide and carbol fuchsin for about five minutes, make sure not to burnt or heat
it to boil as it may cause the death of the colonies and you might not find anything
beneath the microscope. Each time smoke appeared, stop the heating for a while and
continue when the smoke is no longer can be seen. Don’t forget to add more carbol
fuchsin before continue heating.
 After 5 minutes, throw out the excess carbol fuchsin and wash with running water
 After that decolorize the slide using acid alcohol until only a thin layer of red stain is
left.
 Add methylene blue for one minute without any heating needed and then wash with
running water.
 Dry the slide by tapping it with tissue or towel gently and the slide is ready to be
observed under the microscope.

Answer the following questions:

1. What kind of chemical intensifier do you use in the Ziehl Neelsen staining?
Carbol Fuchscin
 2. Why do you need heating in the Ziehl Neelsen staining?
Yes, in order to let the Carbol Fuchsin reside inside cell wall under the wax substance
on bacteria’s surface

3. What will happen if you use alcohol 96% instead of alcohol acid in Ziehl Neelsen
staining?
The stain wil be all washed out and turned transparent because alcohol 96% used as
a decolorizer for excess stain on bacteria surface, meanwhile acid alcohol only
decolorized the stain on non acid fast bacteria and the acid fast bacteria will keep
retain the stain

4. What international standard do you use to interpret the quantity of acid fast bacilli
you find?
AFB evaluation and counting IUAT is the international standard to interpret quantity
Acid Fast Bacilli

Explain what kind of microscopic appearance you expect to find as acid fast bacteria in
Ziehl Neelsen staining result.

(Can be finished before practical work)

I expected a red dots or area on the slide that the Ziehl Neelsen stain method had applied to it

Mention at least two kinds of media that can be used for Mycobacterium tuberculosis
culture
(Can be finished before practical work)
Lowenstein Jensen (LJ) and Middlebrook medium can be used for Mycobacterium
tuberculosis culture
Question Chain 2
Insert the photos you take from DEMO of Ziehl Neelsen stains into the following boxes
and define the photo.
Put arrows and give explanations in the picture to notice your findings!

(Can be finished after practical work)

Acid-Fast Bacteria

Non-Acid-fast Bacteria

Definition
Morphology of acid fast bacteria Bacillus

Color of acid fast bacteria Red

Color of non acid fast bacteria / Blue

 cells

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Note:
Morphology: coccus, bacillus, spiral
Color: red, purple, blue, green, etc
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Insert the photos you take from DEMO of Mycobacterium tuberculosis culture into the
following boxes and define the photo:
(Can be finished after practical work)

Definition
Name of culture medium Lowenstein Jensen

Color of acid fast bacterial Brown

colony

Special characteristic of acid M. tuberculosis appears as brown, granular colonies

fast bacterial colony (sometimes called "buff, rough and tough").

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Note:
Example of culture medium: mannitol salt agar, blood agar, nutrient agar, chocolate agar,
MacConkey agar, eosin methylene blue agar, Lowenstein Jensen, Middlebrook, sabouroud
dextrose agar, thioglycolate, chopped meat/cooked meat, etc. You can add another set of boxes
if you need.
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_________
References:
………………