Chapter Eight
Basic Microbiological Tests
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learning objective
• At the end of this chapter students are expected:-
To define microbiology
To discuss staining, types of staining, and principle of
staining in medical microbiology
Discuss about gram staining and AFB staining
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1. Introduction to Medical Microbiology
– Microbiology is the study of microorganisms, organisms that are too
small to be seen with the naked eye
• Medical Microbiology: - a branch of medical sciences that deals with
microorganisms that cause infectious diseases of human beings.
They include:
o Bacteria (singular, bacterium)
o Viruses (singular, virus)
o Fungi (singular, fungus)
o Protozoa (singular, protozoon)
Based on their size
• Viruses < Bacteria < Fungi< Parasites
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2. Medical Microbiology Specimens
• Specimens are material tested to determine the presence or absence of
specific microbes
• Microbiological specimens consist of:
– Urine - body fluids
– Stool - body tissue
– Sputum
– Wound drainage
– Blood
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3. Microbiological Methods
• The laboratory investigation of microbial diseases involves
isolation & identification of pathogens or their products using:
1. Microscopy
2. Culture techniques
3. Biochemical methods
4. Drug sensitivity testing
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1. Microscopy
There are two principal ways of preparing a microbial specimen
for microscopy.
1. Unstained smears (wet preparation): to examine the
motility of the bacteria.
2. Stained smears: to study the size, shape, arrangement
& staining affinity of the bacteria
Shape
Arrangement
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Microscopy….
Staining:- is the process of the coloring of colorless objects using stains
(dyes).
Bacterial staining:- is the process of imparting color to the colorless
structures (cell wall, spore, etc) of the bacteria in order to make it visible
under the microscope
Uses of staining
a) To observe the morphology of bacteria.
b) To differentiate one group of bacteria from the other group.
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Microscopy….
Basic principle of staining
• Acidic part of the cell is negatively charged (nucleic acid, nucleoproteins
of the nucleus) stains with basic dye (methylene blue), and
• Basic component of the cell positively charged (cytoplasm) stains with
acidic dyes (eosin).
• the structure stained by a combination of these two (neutral stain).
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Preparation of Specimen for Microscopy…
• Procedure: Smear air-dry heat-fix stain
• Smear: to distribute bacterial cells on a slide for staining & viewing under the
microscope.
• Fixation: help the cells adhere to the slide surface. (alcohol or heat)
Staining: three types
1. Simple
2. Differential
3. Structural
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Types of stain
Simple Stains
It involves the use of only 1 dye & is used to study the size,
shape & arrangements of bacteria.
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Types of stain….
Differential Stains
• Multiple stains (dye) are used to distinguish different group of
bacteria
• Types of differential stains
• Gram stain
• AFB stain
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Gram Staining
Purpose:
• Used to study the gram reaction, shape, arrangements & location
of bacteria
• bacteria can be divided into:
Gram positive bacteria: bacteria that retain the
primary stain, crystal violet & so appear purple.
Gram negative bacteria: bacteria that de-stain with
alcohol & appear pink due to counter-stain safranin.
• This difference in staining affinity is due to
difference in the permeability of cell wall called
peptidoglycan.
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Gram Staining….
Required reagents:
1) Crystal violet (Gentian violet).........Primary stain
2) Gram's iodine ................................Mordant
3) Acetone- Alcohol(95% Alcohol).....Decolourizer
4) Safranin(Neutral red).....................Counter stain
Gram staining technique includes the following five major process
sequentially.
o Smear Fix Staining Examining Report
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Gram staining procedure
1) Cover the fixed smear with crystal violet stain for 30–60 seconds.
2) Rapidly wash off the stain with clean water.
3) Tip off all the water, and cover the smear with Lugol’s iodine for 30–60 seconds.
4) Wash off the iodine with clean water.
5) Decolorize rapidly (few seconds) with acetone–alcohol. Wash immediately with clean
water.
6) Cover the smear with Safranin (neutral red) stain for 2 minutes
7) Wash off the stain with clean water.
8) Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry.
9) Examine the smear microscopically,
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Gram Staining….
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Gram Staining….
Interpretation
• Gram-positive bacterium …Purple (Dark purple)
• Gram-negative bacterium …Pink (pale to dark red)
• Yeast cells . . . . . . . . . . . . . . .Dark purple
• Nuclei of pus cells . . . . . . . . Red
• Epithelial cells . . . . . . . . . . . .Pale red
• Report
• Example:-A Gram’s stain of urethral smear report might read as:
• “Moderate number of gram-negative intercellular Diplococci bacteria and many
pus cells seen /HPF
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Ziehl- Neelsen staining Method
• The Ziehl-Neelsen (Zn) technique is used to stain Mycobacterium species.
• Mycobacterium, unlike most other bacteria, do not stain well by the Gram
technique
• Acid fast bacilli (AFB)
Mycobacterium tuberculosis
Mycobacterium leprae
Mycobacterium. ulcerans
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Ziehl- Neelsen staining….
Mycobacterium species
M. tuberculosis and M. ulcerans
• strongly acid-fast.
• 3% v/v acid alcohol solution
• Option:- 25%H2SO4 is used to decolorize the smears.
M. leprae
• weakly acid fast.
• 1% v/v acid decolorizing solution
• Option:- 5% H2SO4
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Ziehl- Neelsen staining….
Reagents required
1) Carbol-fuchsin
2) Acid-Alcohol /H2SO4
3) Methylene blue/Malachite green
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Procedure for Hot Ziehl-Neelson staining method
1)Prepare the smear from the primary specimen and fix it by passing through the
flame and label clearly.
2)Place fixed slide on a staining rack and cover each slide with concentrated carbol
fuchsin solution.
3)Heat the slide from underneath with sprit lamp until vapor rises (do not boil it) and
wait for 5 minutes.
4)Wash off the stain with clean water.
5)Cover the smear with 3% acid-alcohol solution until all color is removed (2-5
minutes).
6)Wash off the stain and cover the slide with 1% methylene blue. for 1-2minute.
7)Wash off the stain with clean water and let it air-dry.
8)Examine the smear under the oil immersion objective to look for acid fast bacilli.
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Ziehl-Neelson staining…
Interpretation
Acid fast bacilli(AFB)…………............Red Rods
• Straight or slightly curved rods, occurring singly or in small
groups
Background material …………….....Blue/ Green
Cells ………………………………………….. Blue/ Green
Background material…………………. Blue/ Green
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Ziehl-Neelson staining…
Reporting of sputum smears
When any definite red bacilli are seen, report the smear as ‘AFB
positive’, and give an indication of the number of bacteria present
as follows:
More than 10 AFB/field………………….+3
1–10 AFB/field ………………………………+2
10–100 AFB/100 fields…………………..+1
1–9 AFB/100 fields ……………report exact number
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Thank you for your atension!!!!!
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