MEDICAL MICROBIOLOGY

Biological Basis of Medicine for

MBBS Degree
By
Associate Prof Dr Priya
Madhavan
School of Medicine
Taylor’s Lakeside Campus
Priya.Madhavan@taylors.edu.my
Ext. 5653
 MEDICAL MICROBIOLOGY

STAINING & MICROSCOPY

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 Learning Outcomes:

1 - OUTLINE THE PRINCIPLES OF

STAINING IN MICROORGANISMS.

2 -IDENTIFY THE SIGNIFICANCE OF

CULTURING MICROORGANISMS.

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MEDICAL MICROBIOLOGY

MICROSCOPY

 Microorganisms & their structural components

are measured in micrometers (µm) or nanometers
(nm).
 Microscopic examination can be done under
different types of microscope.
 Most routine work – light microscopes.

 Detailed/ research work – electron microscopes.

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 (A) ANTONIE VAN LEEUWENHOEK (PRONOUNCED ‘VAHN LAYWENHOOK’)
WAS A DUTCH TRADESMAN AND SCIENTIST FROM DELFT, NETHERLANDS.
HE IS COMMONLY KNOWN AS “THE FATHER OF MICROBIOLOGY.” THE
MICROSCOPE HE DEVELOPED WAS USED TO VIEW ALL KINDS OF CELLS,
MAGNIFIED TO 300 X.

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MEDICAL MICROBIOLOGY

TYPES OF MICROSCOPES
 LIGHT MICROSCOPY
- Use of any kind of microscope that uses
visible light to observe specimens.

 ELECTRON MICROSCOPY
- Use of electron microscopes to observe
objects smaller than 0.2µm.

**What are the differences between light microscopes

and electron microscopes? 6
**Name the types of light and electron microscopes.
7
CLINICAL SAMPLE COLLECTION
Type or Location
Skin, eye, outer ear, nose,
throat, vagina, cervix,
urethra, open wounds
Blood
Cerebrospinal fluid
Collection Method
Sterile swab brushed
across the surface (not to
contact neighbouring
tissues)
Needle aspiration from
vein (to add anti-
coagulants in the
specimen transfer tube)
Needle aspiration from
sub-arachnoid space of
spinal column
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CLINICAL SAMPLE COLLECTION

Type or Location Collection Method

Stomach Intubation – inserting a
tube into the stomach via
nostril.
Urine Aseptic collection - A
catheter is inserted into
the bladder via urethra.
Clean catch – midstream
urine is collected.
Lungs Collection of sputum –
dislodged by coughing or
acquired via a catheter.
Diseased tissues Biopsy – surgical removal.

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Intubation

Urine collection

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Biopsy
 STAINING TECHNIQUES

 Dyes are used to increase contrast between cells

& its background.
 Dyes – organic compounds & have the affinity to
differentiate cellular materials.
 Cationic dyes (Basic) – positively charged ions;
Methylene blue, crystal violet, malachite green,
carbolfuschsin, safranin
 Anionic dyes (Acidic) – negatively charged ions;
Eosine, acid fuchsin, rose bengal, Congo red
 Negative staining – prepare colourless bacteria
against a dark background; Indian Ink, Nigrosin
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MEDICAL MICROBIOLOGY

SMEAR PREPARATION TECHNIQUE

 Before cells are stained, they must be fixed to a

slide.

 Fixing will kill the cells & attach them to the

slide.

 It also preserves various part of the microbes in

their natural state.

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 THE PROCESS …

 A thin film of cells is spread on a slide (smear).

The smear is allowed to air dry OR


 Passing the slide through the flame of a Bunsen burner.

 Stain is applied, leaving for several minutes.

 Stain is washed off.

 Ready for microscopic examination.

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Watch here - https://www.youtube.com/watch?v=MiGp8buyzXg
MEDICAL MICROBIOLOGY

STAINING TECHNIQUES
 Microbiologists use 3 kinds of staining techniques:

(I) Simple stains – an aqueous/alcohol

solution of single basic dye.
(II) Differential stains – react differently with
different types of bacteria.
(III) Special Stains – to colour and isolate
parts of microorganisms, such as
endopores, capsule & flagella.

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MEDICAL MICROBIOLOGY

DIFFERENTIAL STAINING

(A) Gram Stain

 Developed by Hans Christian Gram, 1884.
 Classifies bacteria into Gram positive & negative.
 Crystal violet (primary stain), Iodine (mordant), Alcohol
(decolourlizer), Safranin (counterstain) are used.
 Purple dye-iodine complex will stain Gram positive & negative
cells.
 Alcohol will remove the dye from Gram negative cell wall –
colourless.
 Safranin is added and stains the Gram negative cells pink/red.

Watch here - https://www.youtube.com/watch?v=AZS2wb7pMo4

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GRAM STAINING TECHNIQUE

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GRAM POSITIVE & NEGATIVE CELLS

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MEDICAL MICROBIOLOGY

DIFFERENTIAL STAINING

(B) Acid-Fast Stain (Ziehl-Neelsen stain)

 Binds only to bacteria that have waxy material in their cell walls
(Eg. Species of Mycobacterium & Nocardia).
 Red Carbolfuchsin applied to smear & heated.

 Washed with water & treated with acid-alcohol (decolouriser).

 Red colour is removed from non acid-fast bacteria.

 Acid-fast bacteria retain the red colour because the dye is

lipid soluble.
 Methylene blue (counterstain) is applied & the non acid-fast
bacteria are stained blue.

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ZIEHL-NEELSEN STAINING TECHNIQUE
(ACID FAST)

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APPLICATION OF ACID-FAST STAIN
 Mycobacterium tuberculosis, the bacterium that
causes tuberculosis, can be detected in specimens based on the
presence of acid-fast bacilli.
 Often, a smear is prepared from a sample of the patient’s
sputum and then stained using the Ziehl-Neelsen technique.
 If acid-fast bacteria are confirmed, they are generally cultured to
make a positive identification.

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MEDICAL MICROBIOLOGY
ACID-FAST STAINING

Nocardia sp., Mycobacterium tuberculosis, Mycobacterium

leprae, Mycobacterium kansasii, Mycobacterium marinum, 21
Mycobacterium bovis, Mycobacterium africanum and members
of the Mycobacterium avium.
MEDICAL MICROBIOLOGY

SPECIAL STAINING
(A) Negative Staining

 To observe the presence of capsules.

 Mixing cells with a colloidal suspension of coloured particles
(Indian Ink/ Nigrosin).
 This would provide a dark background.
 A simple stain (safranin) is applied.
Capsules of
 Capsules don’t accept dyes & appear as clear halos Bacillus
surrounding each stained cell. anthracis

Capsules of
Cryptococcus
neoformans

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MEDICAL MICROBIOLOGY

SPECIAL STAINING
(B) Endospore Staining

 Uses an endospore stain called Schaeffer-

Fulton stain.
 Malachite green (primary stain) applied to

heat-fixed smear & steamed for 5mins.

 Washed off with running water to remove

dye from cell.

 Safranin added to stain the cell.

 Endospores appear green within pink/red

cells. 23
ENDOSPORE STAINING

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MEDICAL MICROBIOLOGY

SPECIAL STAINING
(C) Flagella Staining
 Flagella is too small

to be seen under the

microscopes without
staining.

 Uses a mordant
(tannic acid or
potassium alum),
then stained using
either pararosaline or
carbolfuchin to build
up the diameter of 25

the flagella.
 SELF-STUDY QUESTIONS

 Explain why it is important to fix a specimen before

viewing it under a light microscope.
 What types of specimens should be chemically fixed as
opposed to heat-fixed?
 Why might an acidic dye react differently with a given
specimen than a basic dye?
 Explain the difference between a positive stain and a
negative stain.
 Explain the difference between simple and differential
staining.
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MEDICAL MICROBIOLOGY

STERILISATION TECHNIQUES

 Aseptic technique – complete sterilisation technique to

avoid all contaminants.
 Microorganisms are microscopic & are everywhere.

 All culture medium must be sterilised soon after

preparation.
 When containers are opened, it must be handled in a
way to avoid laden air from entering.

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MEDICAL MICROBIOLOGY

PRINCIPLES OF STERILISATION
 Kill microbes by denaturing their enzymes.
 Degree of heat resistance of bacteria must be
considered (Thermal Death Point).
 Length of time to be rendered sterile (Thermal Death
Time).
 Time taken for 90% of bacterial population to be killed
at a given temperature (Decimal Reduction Time)
 2 types of heat sterilisation – dry heat & moist heat.
 Dry heat – Flaming, Incinerating, Hot air.
 Moist heat – autoclaving, boiling, pasteurisation.

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MEDICAL MICROBIOLOGY

PASTEURISATION
 Louis Pasteur found a
practical method of
preventing spoilage of
beer & wine (1863).
 He used mild heating –
applied to sterilisation of
milk later on.
 Dairy industries usually
use indicators to
determine the sterility of
products.
***How does pasteurisation
control microbial growth?
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MEDICAL MICROBIOLOGY

OTHER METHODS
(A) FILTRATION
 To sterilise heat-sensitive materials – culture
media, enzymes, vaccines, antibiotic solutions.
 HEPA (high efficiency particulate air filters)
remove almost all microbes larger than
0.3µm in diameter. E.g. in operating theaters.
 Now, membrane filters are used – 0.1mm thick
cellulose esters/ plastic polymer filters which has
pore sizes of 0.01µm to 0.45µm.

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MEDICAL MICROBIOLOGY
Membrane Filtration

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Watch here - https://www.youtube.com/watch?v=B1AVxzccS5Q

MEDICAL MICROBIOLOGY

OTHER METHODS

(B) LOW TEMPERATURES

 Depends on particular microbes.

 0-7°C refrigerator T would reduce the metabolic

rate of most microbes.

 Some bacteria can grow below freezing T.

 Many parasites (roundworms) are killed within

several days after freezing.

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MEDICAL MICROBIOLOGY

OTHER METHODS
(C) DESICCATION
 In absence of water, microbes are unable to

grow.
 Varies with species.

 Viruses are usually resistant to desiccation.

 Dust, clothing, bedding, dressings may

contain microbes in dried pus, blood, urine,

mucus.
 Some bacterial sp can only withstand
dryness for an hour (Neisseria gonnorhoea)
& others maybe for months (Mycobacterium 34
tuberculosis).
MEDICAL MICROBIOLOGY
TECHNIQUES TO CULTURE MICROORGANISMS
Learn these terminologies:
 Culture medium – an aqueous solution of various
nutrients suitable for the growth of microorganisms.
 Culture – a particular strain or species of organism
growing in a laboratory medium.
 Pure culture – a culture containing a single kind of
microorganism.
 Mixed culture – a culture containing two or more
kinds of microorganisms.
 Inoculum – a suspension of microorganism in a liquid
medium or sterile water.
 Plating – transferring microorganisms from a culture
onto an agar plate.
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MEDICAL MICROBIOLOGY

ASEPTIC TRANSFER
 To transfer from one
tube to another using
an inoculating loop or
needle.
 To transfer culture to
an agar plate by
streaking from an
isolated colony.
 To obtain a pure
culture from a
microbial colony. 36
37
MEDICAL MICROBIOLOGY

VARIOUS CULTURE METHODS

 Broth Culture – organisms grown in liquid
media.

 Slope Culture – organisms grown on slant

agar in test tubes or small bottles.

 Stab Culture – organisms grown by stabbing

vertically into solid or semi solid medium
(gel) in test tubes or bottles.

 Plate Culture – organisms grown by

streaking on agar plates in Petri dishes.
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 SLANT AGAR & SOFT AGAR (STAB
CULTURE)

Preparing a slant … Stab cultures …

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MEDICAL MICROBIOLOGY

STREAK PLATE TECHNIQUE

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MEDICAL MICROBIOLOGY

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MEDICAL MICROBIOLOGY

0BTAINING A PURE CULTURE

 To obtain a pure culture from a mixed culture using
streak plate method:

1- With a sterile loop, transfer a single colony from the

original plate onto a new agar plate.
2- After several days, check for growth.
3- If the plate has one type of microorganism (compare
colour, shape, size, growth pattern), then a pure
culture is obtained.
4- If plate has more than one type, repeat the steps
until only one type of microbial growth occurs.
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MEDICAL MICROBIOLOGY

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MEDICAL MICROBIOLOGY

MIXED CULTURES ON AGAR

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COLONY MORPHOLOGY

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DESCRIBING AND REPORTING COLONY
MORPHOLOGY

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 PROPER IDENTIFICATION OF PATHOGENS
Clinical specimen
collection

Culture on agar
Stain
plates

Observe colony Observe cell

morphology morphology under
microscope

Biochemical tests,
Serology tests
Molecular identification 47
MEDICAL MICROBIOLOGY

PRESERVING MICROORGANISMS
 Short term – 4 to 7 days; refrigeration on agar slants,
Petri dishes, liquid medium.
 Long term – several years; deep-freezing & freeze-
drying.
(i) Deep-freezing – pure culture is suspended in liquid
medium & quickly frozen at T btw - 50°C to - 95°C.
(ii) Freeze-drying – a suspension of microbes quickly
frozen at T btw - 54°C to - 72°C; water is removed by
vacuum.

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MEDICAL MICROBIOLOGY

STUDY QUESTIONS

1. Compare and contrast the techniques and usage of

different staining methods.

2. Compare and contrast the effectiveness of dry heat

and moist heat sterilization techniques.

What mordant is used in Gram staining?

a. crystal violet
b. safranin
c. acid-alcohol
d. iodine
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MEDICAL MICROBIOLOGY

STUDY QUESTIONS
3. Why isn’t the Gram stain used on acid-fast bacteria?

4.Discuss the relationship of the cell wall structure to

Gram staining.

5.Discovery of endospores was of great practical

importance. Explain why.

6.How could you identify whether a particular bacterial

sample contained specimens with mycolic acid-rich cell
walls?
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The End of Topic

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