Microbiology – Practical syllabus

1. Microscopic Principles and Applications

A microscope consists of:

- Objective lens
- Ocular lens
- Lens tube
- Stage
- reflector

The magnification of the microscope is the product of the objective and ocular lens magnification.

- Resolution is the microscope's ability to identify to light spots separately, and the shortest distance
between features that can be identified.
- In effect, resolution determines how clearly something is seen.
o The highest theoretical resolution is 100 to 200 nm due to effect of wavelength.
- All lenses also have a numerical aperture which illustrates the resolution and brightness of the lens.

Microbes (the subject of microbiology) are microscopic, that is to say, smaller than a human eye can see, as
such we must magnify. On average, microbes tend to be 1/10th the size of a typical human cell.

Applications include  Loeffler’s stain, gram stain, Neisser stain.

2. Loeffler’s stain
Background

Microbiological staining can be separated into 2 types: simple and compound

- Simple  uses one dye; the size, shape and distribution of bacteria can be seen.
- Compound  uses more than one dye; the size, shape, distribution and some extra details of the
bacteria can be seen.

Loeffler’s stain is a type of simple staining that uses methylene blue. Another type of staining, Pfeiffer’s stain,
uses the same method as Loeffler’s except it uses fuchsin as a dye, so the microorganisms and cells will
appear red instead of blue.

Method

- Prepare a fix a glass slide with a sample

- Add methylene blue onto the surface of the slide and let it rest for 2-5 minutes
- Wash the slide with water and allow it to dry at room temperature.
- Examine the slide under an oil-immersion objective (x100).

3. Gram stain – preparation, observation and interpretation

Background

Gram staining is a type of compound stain that differentiates microorganisms into gram-positive and
negative on the basis of differences of cell wall structure. This is done for the diagnostic identification of
bacteria.
Microbiology – Practical syllabus

Both gram positive and negative organisms have a cell wall which is a layer outside of the bacterial
cytoplasmic membrane made up predominantly of peptidoglycan.

- Gram positive bacteria  stain blue/violet with this method. This is because the stain gets trapped
within the thicker, mesh-like peptidoglycan layer.
- Gram-negative bacteria  stains red with this method. These bacteria have a thinner peptidoglycan
layer that does not retain the gentian violet stain, so the stain is replaced with fuchsin when it is
added and will turn red.

Mycoplasmas have no cell wall, and hence no peptidoglycan, so they cannot be stained by this method.

Method

- Prepare and fix a glass slide

- Add gentian violet to the sample and leave to dye for 2-5 minutes. Pour off
- Add Lugol’s iodine solution and leave to dye for 1-2 minutes. Wash off with water
- Add 95% ethanol for 15-20 seconds to wash the gentian violet; the ethanol acts as a decolouriser.
- Wash the preparation with water
- Add fuchsin for 1-2 minutes. Wash off with water. Allow it to dry at room temperature
- Examine using a x100 oil-immersion objective.

4. Neisser stain – preparation, observation and interpretation

Purpose

This method stains the volutin (or “metachromatic”) granules of Corynebacterium diphtheriae, Lactobacillus
etc. Bacterial inclusions such as volutin bodies act as a reserve for nutrient substances.

This method is a type of compound stain that allows observation of size, shape and distribution of bacteria,
as well as volutin inclusion bodies. These inclusion bodies are made up of products stored within specific
granules and will stain brown-violet with this technique.

Method

- Prepare and fix a glass slide

- Add Neisser 1 solution to the sample and leave to dye for 2-3 minutes. Pour off
o Neisser 1 is a solution of Neisser A (acetic acid solution of methylene blue) and Neisser B
(gentian violet) in a ratio of 2:1
- Add Neisser 2 solution and leave to dye for 2-3 minutes. Pour off
o Neisser 2 is a solution of chrysoidine)
- Wash the slide with water, allow to dry
- Examine using a x100 oil-immersion objective

Result

Volutin granules appear dark blue, and the cytoplasm appears yellow.

Corynebacterium diphtheriae are visualised as rods that contain dark blue granules; the granules have a high
concentration of poly-metaphosphates with acid character and a greater affinity to alkaline dyes, which
leads to a more intense dark blue stain in both ends of the rod vs in the cytoplasm.
Microbiology – Practical syllabus

5. Smear preparation and staining by Ziehl-Neelson method. Technique,

observation and examination of the preparations.
Purpose

Ziehl-Neelsen staining is a type of compound staining method; acid-fast bacteria that cannot be classified by
gram stain are distinguished by this method. This includes mycobacteria, which have a waxy outer shell.

- Includes mycobacterium tuberculosis and leprae (causes leprosy)

o Most specimens are respiratory; e.g. early morning sputum.

The thick waxy coating that covers these bacteria are resistant to staining, however once they stain, they are
resistant to decolourisation by acid and alcohol.

- Acid-fast organisms resist decolourisation by acid alcohol; the cell wall lipids of the Mycobacteria do
not dissolve when the acid alcohol is applied, hence the red stain remains.

Method

- prepare a smear directly from sputum; take sample from sputum using a loop and spread it over a
glass slide to form a thin film
- air-dry and fix the slide, then flood it with carbolfuchsin stain
- heat the slide 3x with a burner until vaporisation occurs; do not allow it to boil. Let the solution cool
and allow the dye to work for 5 minutes
- wash the slide with water, then with 5% H2SO4 for 1-2 mins
- remove the sulphuric acid and wash the slide with ethanol for 1-3 minutes
o the acid and ethanol act as decolourisers.
- Wash with water, then add methylene blue. Allow to dye for 1-2 minutes
- Wash with water and air-dry. Examine using a x100 oil-immersion objective

The acid-fast bacteria will stain red, whereas non-acid-fast will stain blue.

6. Smear preparation and staining by Peshkov method. Technique, observation and

examination of the preparations.
Background

Some gram-positive (but not gram-negative) bacteria, such as Bacillus and Clostridium, can form spores.
These are dehydrated, multishelled structures that allows the bacteria to exist in unfavourable conditions;
bacteria are converted from a vegetative state to a dormant state.

The location of a spore within a cell is a characteristic of the bacteria that can assist in its identification.
Endospores are very resistant to staining by simply dyes until they germinate; dormant endospores, once
stained, are resistant to decolourisation.

- Centrally or sub-terminally located spore with no swelling of the cell is indicative of the Bacillus
species
- Subterminal spore location with cell swelling is in most Clostridium species
- Terminal spore location with cell swelling is Clostridium tetani and other Clostridium species
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Method

- Apply some isotonic saline or water onto a clean glass slide

o No need for the saline if the sample is a liquid
- Sterilise the loop, then allow it to cool and take a sample from an isolated culture of Bacillus cereus
grown on a solid medium
- Using the loop, spread the sample onto the saline/water on the glass slide and create an area of 1-
1.5cm diameter. Sterilise the loop again
- Air dry and fix the glass slide using the burner
- Apply methylene blue to the slide and heat it until it boils. Wash the dye off with water
- Apply 0.5% aqueous solution of neutral red for 1-2 minutes. Wash off with water
- Allow the slide to dry and examine using a x100 oil-immersion objective

Result: The endospores should appear blue, and cytoplasm of vegetative cells will appear rose.

7. Cultural diagnosis of bacterial diseases

Background

A culture is a way of growing bacteria (or other microorganisms) in a laboratory setting; the characteristics of
the colonies grown on a culture can be used to identify a specific type of bacteria. Cultures are contained on
plates or tubes that contain specific media that allows a particular pathogen to grow.

Bacteriological media should be sterile, isotonic, pH adjusted, and inoculated with nutrients that allow for
optimal bacterial growth. They are usually left for a few days at either room or body temperature before
being examined to allow colonies to form.

Bacterial growth on an agar plate provides some information about:

- Colonies  size, shape, colour/pigmentation

- Haemolysis:
o Alpha  green discolouration around colonies and partial lysis of RBCs; typical of S.
pneumoniae
o Beta  clearing of blood agar around the colony due to complete lysis of RBCs typical of S.
aureus and S. pyogenes
o Gamma  no lysis of RBCs; colonies look normal because no haemolysis occurred. This is
typical of S. epidermidis.
- Smell  characteristic for some species

Culture media can be categorised according to:

- Consistency  broth, semi-solid, solid agar

- Composition  common or special (enriched)
- Purpose  non-selective; selective; elective; differential; anaerobic.

(Give examples using the next few points about cultures).

Microbiology – Practical syllabus

8. Simple nutrient media. Types, composition and application. Observation

(characteristic) of bacterial growth in broth and agar media.
Background

Cultivation is the process by which microorganisms are given the appropriate conditions, nutrients and other
factors to allow them to grow and multiply in an artificial media. In order to grow, bacteria will need:

- Environmental factors  moisture, warmth (37C), oxygen (in some cases), CO2, pH balance
- Macroelements  N, K, H, Ca, S, C, P
- Microelements  Fe, Cu, Mn, Cl, Na

These factors are all provided by a simple nutrient media whose purpose is to non-selectively grow most
bacteria. E.g. meat peptone water, peptone gel, peptone agar. Addition of supplements such as blood, GFs
and antibiotics will turn a simple media into a special media.

A bacterial cell that is left in an ideal condition for a specific amount of time will eventually begin to divide.
After 18-24 hours, a colony will be formed that is visible with the naked eye. These colonies have specific
sizes, shapes, colours, peripheries/centres and smells.

(Compare with special media using point 9 info).

9. Special (enriched) nutrient media. Types, composition and application.

Observation (characteristic) of bacterial growth in broth and agar media.
Background

An enriched media is created when supplements are added to a basic media.

Elective media, such as blood agar or chocolate agar, are used promote the growth of a desired bacteria,
without inhibiting the growth of other species. Blood agars are a type of growth medium that encourages
growth of certain bacteria, such as streptococci, that would not otherwise grow.

- Blood agar is the most commonly used media in microbiology; created by adding blood to a simple
agar. On these blood cultures, bacteria can be differentiated into:
o Alpha haemolytic  bacteria partially destroy erythrocytes; some are used for their
metabolism. E.g. Streptococcus pneumoniae. Green discolouration is created around the
colony
o Beta-haemolytic  all erythrocytes beneath and around the colonies are destroyed. E.g.
Staphylococcus aureus, Streptococcus pyogenes
o Gamma-haemolytic  no haemolysis; these bacteria are not pathogenic to humans
- Chocolate agar is named so because of the brown colour; it is an example of blood agar that is
heated up until erythrocytes are destroyed and haem is released.
o The bacteria that grow on this medium are those that are slower metabolisers and need an
already destroyed medium instead of destroying it themselves.
o E.g. Neisseria, Enterococcus, Haemophilus influenzae

Selective media are created by adding preferred substances or inhibitory agents to a simple media in order
to facilitate the isolation of a desired bacteria and inhibit the growth of other species.
Microbiology – Practical syllabus

- E.g. antibiotics, dyes and other substances can be used as selective agents.
- Bile salts are an example of an inhibitory agent in apocholate citrate agar for enteric pathogenic
bacteria.
- PPLO (Mycoplasma) agars and broths are used for isolating and cultivating Mycoplasma

Differential media are used to differentiate bacteria according to the products that they produce at the end
of a metabolic process. These products are highlighted by indicators adding to a media, which can cause the
media to change colour.

- E.g. MacConkey agar is used for differentiation of enteric bacteria into lactose positive or lactose
negative; production of acid due to lactose metabolism reacts with an indicator to produce a colour
change (usually from yellow (-) to red (+)).
o Bacteria that are “lactose negative” lack the enzyme that produces the product that reacts
with the indicator to produce the change in colour.

Transport media are used after taking a sample from a patient in order to preserve the microorganism until
it is transported from the ward to the lab. E.g. Stuart media

10. Methods and nutrient media for cultivation of aerobic and micro-aerophile
bacteria. Preparation of pure culture
Cultivation of aerobic and micro-aerophile bacteria

Bacteria differ in their atmospheric requirements for growth.

- Aerobic  grow in oxygen

- Micro-aerophilic  require strictly limited oxygen due to having no electron transport system.
- Facultative anaerobes  can grow in aerobic or anaerobic conditions;
o these bacteria use O2 as an electron accept and have catalase and superoxide mutase, but
they can also grow with no O2 via fermentation

Preparation of a pure culture

Pure cultures are used as a source of identification and susceptibility testing of bacteria. These are obtained
from isolated colonies, since one colony arises from a single bacterial cell.

To grow a colony in liquid media: use a sterilised loop to take the sample

- spread the sample on the inner part of the tube, then mix it gently with some of the liquid media
and put it in the tube. Do not immediately place it directly in the media
- cultivate for 24hrs in 37C

To grow a colony in solid media, a tube or petri dish can be used

- Tube  using a sterilised loop, take the sample

o Horizontal line  make a hole in the gel using the needle, without touching the bottom of
the tube, then twist and remove the needle from the gel
o Sloped line  do the same, then spread the sample over the surface of the gel
- Petri dish  use a loop to spread the sample roughly first
o Sterilise and spread in parallel stripes
o Sterilise and spread in perpendicular stripes
Microbiology – Practical syllabus

o Sterilise and spread diagonally

o Sterilise then make a wiggle
o This method decreases the concentration of organism in each quadrant of the dish

Once the colonies have been cultivated, one is selected to grow on another agar plate. Using a sterilised
needle, remove the central part of the colony (or the whole colony, depending on its size) and inoculate the
agar media as described above.

Pure cultures can be used for:

- Glass slide and staining, Antibiogram

- Biochemical tests, Serology tests, DNA/RNA tests

11. Methods and nutrient media for cultivation of anaerobic bacteria. Reading the
growth of anaerobe bacteria
Background

Obligatory anaerobic bacteria cannot grow in the presence of air. They can be stimulated by:

Chemical methods  gas pack system: petri dishes containing bacteria re placed into a box nearby chemical
reagents and then the box is sealed to prevent new air from entering it

- Chemical reagents use up the residual oxygen, creating an oxygen-free atmosphere to allow the
anaerobes to grow.

Biological method  a petri dish is used and the central area of the gel is cut out using a sterile knife. On
one side of the gel are facultative anaerobes, on the other are obligatory anaerobes

- The dish is closed to prevent oxygen from entering and sealed with paraffin for reassurance
- After cultivating at 37C for 24hrs, the facultative anaerobes will have used up all of the remaining
oxygen, allowing the obligatory anaerobes to grow in absence of oxygen.
- This is called the Fortner technique

Physical methods  evacuation-replacement system; a jar is evacuated, washed with nitrogen, evacuated
again, then filled with an oxygen-free gas mixture (80:12:8 = N2:CO2:H2).

- Must be used with a palladium catalyst for reducing conditions

Selective media  uses Komkova, Shedler or Taroci liquid media. These contain muscle and liver tissue at
the bottom of a tube.

- The liquid media should be boiled for 15 minutes and immediately cooled and inoculated.
- Then, liquid paraffin is added to the top of the tube, sealing it and preventing new oxygen from
entering.
- The oxygen that is already in the media is used up by the tissues in the media
- Cultivate and add the pure culture; the media has no oxygen so bacteria can grow.
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12. Biochemical tests for the identification of bacteria. Tests for the determination
of carbolytic, proteolytic enzymes and oxidoreductases. Other tests.
Tests for carbohydrate metabolism

Bacteria that contain enzymes that act on sugars will usually produce some acid as a product. An indicator
can be used to react with the acid and produce a change in colour, indicating that the bacteria is able to
metabolise sugar. To analyse sugar usage by bacteria:

- Prepare several tubes; Hiss media is a nutrient medium with a neutral pH

- Add different sugars in each tube; glucose, lactose, sucrose, mannitol etc.
- Add a pure culture. Allow to cultivate for 24hrs at 37C
- On the next day, add Andrade’s indicator (acid fuchsin neutralised by NaOH, which is yellow)
o The indicator is yellow. A positive test is dark pink
o Colour change to dark pink indicates a change in pH of the medium to 5.5, which
demonstrates that an acid has been produced after enzymes acted on the sugars

Metabolism of proteins and amino acids

Indole test  demonstrates the ability of bacteria to decompose the amino acid tryptophan into indole.
Hydrogen sulphide can also be produced. No products are produced if the bacteria does not have the
appropriate enzymes

- Add trypsin broth to test tubes

- Add a pure culture; cultivate at 37C 24hrs
- Add 0.5mL Kovach’s reagent as an indicator
o A red colour in the surface layer of the tube indicates an “indole positive” result
o If negative, the colour stays yellow

Phenylalanine deaminase test  indicates the ability of an organism to deaminate phenylalanine to produce
pyruvate, which does not have a colour. Pyruvate is reacted with ferric salts to produce a green colour.

- Inoculate and incubate for 24 hrs at 37C

- Add a few drops of 10% ferric chloride solution to the edge of the tube to allow it to run down over
the growth on the slope.
o Green is a positive result. Staying yellow is a negative result

Test for detection of oxidative/reductive enzymes

Catalase test  demonstrates the presence of the enzyme catalase, which catalyses the reaction of [H2O2
 H2O and O2]. The test can be performed on a glass slide or in a test tube.

- A small amount of a culture to be tested is taken from a nutrient agar using a sterilised loop and
added to a 3% H2O2 solution.
o Production of bubbles indicates a positive result (O2 produced)
o Staphylococcus is catalase positive; Streptococcus is catalase negative.

Oxidase test  determines the presence of the bacterial cytochrome enzyme oxidase. Cytochromes, in
aerobic respiration, transfer electrons to oxygen to form H2O.

- A filter paper is placed over a glass slide. The paper contains a reagent called TMPD, which is a dye
that acts as an artificial electron acceptor, substituting oxygen.
Microbiology – Practical syllabus

- In presence of the enzyme cytochrome oxidase, the bacteria transfers electrons to the filter paper
and causes it to change colour.
o Purple is a positive result, indicating that TMPD radical has formed by the transfer of
electrons by the enzyme.
o TMPD = tetramethyl-p-phenylene-diamine dihydrochloride.

Urease test  the presence of urease is tested for by growing an organism in Christensen medium in
presence of urea. Ammonia is produced as a result of urea metabolism.

- Ammonia changes the pH of the medium to become more basic; phenol red is used as a pH
indicator. A change of colour to pink indicates a positive result.
o Christensen medium is initially yellow

Other tests

Citrate test  tests the ability of a microorganism to utilise citrate as a sole carbon source. Uses a solid
media called Simmon’s citrate agar, which contains sodium citrate as the sole carbon source, and
ammonium phosphate as a nitrogen source

- Bromthymol blue dye is added as an indicator; it is green at pH 6.9 and blue at pH 7.6
- Inoculate from a saline suspension of the organism to be tested
- Incubate for 24hrs at 37C
o A positive reaction is blue, a negative reaction is green

13. Kligler’s polytrope medium (Triple Sugar Iron Agar): composition, inoculation
and reading.
Purpose

Kligler’s iron agar is used to perform 5 tests at the same time in the same tube; the tube is separated into 2
areas; above and below the slope:

- The area below the slope checks for glucose usage

- The area above the slope checks for lactose usage
- The middle area checks for production of hydrogen sulphide
- The hole produced by the needle is used to check for urease activity
- The entire agar is used to check for gas production (bubbles)
Microbiology – Practical syllabus

A negative result is when all parts of the agar remain orange and no bubbles are seen.

A positive result indicate that the bacteria have enzymatic activity that uses the substrates; in example D, the
results for glucose and urease cannot be seen because the indicator for H2S blocks it. In this scenario,
individual tests can be performed.

- Lactose  colour change above the slope from orange to yellow; indicative of beta-galactosidase
activity
- Glucose  colour change below the slope from orange to yellow
- H2S  colour change to black
- Urease  colour change to pink
- Bubbles  indicates production of gas; appears as fissures in the medium. Gases are produced as a
result of carbohydrate fermentation

Kligler’s agar is primarily used to differentiate members of Enterobacteriaceae and to distinguish them from
other Gram-negative bacilli such as Pseudomonas or Alcaligenes

14. Quantitative methods for the determination of bacterial growth: enumeration

with optical standard and determination of microbial count on a solid medium.
Bacterial enumeration

Bacterial count is the number of bacteria tested in a volume of 1mL. several methods for determination of
bacterial count are used:

Microscopy  for example, used in examination of urine in clinical microbiology

- Add 50uL of urine to a flat-well microtitre plate

- Add 50uL of diluted toluidine blue
- Cover each filled row of the tray with cellotape. Gently mix the wells
- Wait 5 minutes for the cells to settle
- Examine with a Wilovert inverted microscope using the x20 objective first
- Count the number of cells of each type within the hole area of the graticule

Turbidimetry  counts all viable and dead bacterial cells. A solution of bacteria is inserted into a machine
that has a structured tunnel of a specific diameter;

- The diameter of each individual bacteria are sized in that they can pass through the tunnel one at a
time only.
- A light is shone onto the tunnel; when it hits a bacterial cell, the light rays disperse on the other side
of the cell (1 ray disperses into 3 rays)
- The amount of light rays produced is recorded and used to enumerate the number of bacteria in the
solution. This method is thought to be very precise

Optical standard  this method is less precise but still useful in clinical microbiology. Tubes of microbial
cultures, which all have varying densities, are compared against the optic standard known as MacFarland
standard.

- 0.5 MacFarland units is representative of 3.5x10^8 bacteria in 1mL

o Standards of 0.5, 1, 2 etc until 5 MacFarland units are used
- The amount of bacteria in a tube is determined by comparing a test tube against the standard
Microbiology – Practical syllabus

10-fold dilution method  used for determination of viable bacteria count; commonly used to test the
amount of bacteria in drinking water.

- Prepare 7 tubes; add 0.9mL saline to each tube.

- Add 0.1mL of the sample to test (e.g. drinking water) to the first tube; this dilates the amount of
bacteria in the sample by 10x
- Perform a serial dilution; 0.1mL from tube 1 is added to tube 2, then 0.1mL from tube 2 into tube 3
and so on.
o 0.1mL is discarded from tube 7 to ensure each tube has the same volume of solution
o Tube 1 has the highest concentration of bacteria and tube 7 has the lowest concentration.
- From tubes 5, 6, and 7, 0.1mL solution is taken and cultivate on a petri dish at 37C for 24hrs.
o Colonies will have formed by the next day with most colonies forming on the dish containing
sample from tube 5, and the least from tube 7.
o A formula is used to determine the bacteria count on each petri dish according to the
number of colonies that formed:
 Bacterial count = number of colonies x reverse dilution x 10
o Reverse dilution  e.g. if the dilution of the respective tube was 10^-6, the reverse dilution
will be 10^6.
o The formula is multiplied by 10 because 0.1mL is used each time and the number of bacteria
is supposed to be counted according to 1mL (correction for units)

15. Microbiological examination of drinking water, soil, air, foods. Methods and
interpretation of the results
Water

The parameters of water quality are:

Physical Turbidity, palatability, conductivity, total dissolved solutes

Chemical pH, dissolved O2 concentration, residual free chlorine, radionuclides,
organic/inorganic chemical contents
Microbial Faecal coliform count, total coliform count, faecal streptococci, cysts/ova of parasites

The Colilert test is a commercial preparation that examins drinking water for the presence of total coliforms
(in general) and E. coli (specifically).

- Colilert reagent is formulated to favour the growth of coliforms and inhibit that of non-coliforms. It
is added to a clear bottle containing a 100mL sample of drinking water.
Microbiology – Practical syllabus

o The sample is incubated at 35C for 24 hours and then compared to a control.

All coliforms ferment lactose using the enzyme beta-galactosidase. Colilert reagent contains an indicator
called galactopyranoside, which is an artificial substrate of the same enzyme. when it is hydrolysed, it
produces a yellow-coloured product (o-nitrophenol).

- If the test bottle is as yellow or more yellow than the control, this is a positive result: reported as
“total coliforms present” in the water sample.

The enzyme beta-glucuronidase is present in E. coli; Colilert reagent contains an indicator called MUG (4-
methylumbelliferal-beta-D-glucuronide) which acts as a substrate for this enzyme.

- A positive result produces fluorescence when viewed under a UV lamp; this indicates the presence
of E. coli in the water sample.

A negative result for both these tests is if the water sample looks the same as the control sample.

Food, air, soil

Food  immunoassay, PCR, culture (not suitable for all food groups)

Air  petri dishes are kept open containing agar medium for a measured period of time. Eventually, large
bacteria-containing dust particles will settle on the medium and begin to cultivate. The plates are incubated
and the colonies formed are counted.

Soil  microscopic examination, contact slide method, soil enrichment method

- Contact slide method  a glass slide is sterilised and inserted into a sample of soil that is then
firmed up around the slide for maximum contact and left for 1-3 weeks.
o The slide is then wiped with a paper towel and can then be stained and observed under a
microscope.

16. Serial Dilution Method for Antimicrobial Susceptibility Testing

Minimal inhibitory concentration

MIC represents the minimal concentration of antimicrobial agent that is required to inhibit the
microorganism; expressed in ug/mL. Antimicrobial agents are tested at a two-fold serial dilution and the
lowest concentration that inhibits visible growth is recorded as the MIC.

- Prepare around 12 tubes; add 1mL of saline to all tubes

- Add 1mL of an antibiotic of known concentration to tube 1; the antibiotic has been diluted 1:2.
- Perform a serial dilution in all tubes except for the last, which will be used as a control. 1mL from the
tube before the last one is taken and discarded
- Add the tested pure culture to all tubes. Allow to cultivate at 37C for 24hrs.
o More bacterial growth will be seen in tubes that have a lower concentration of antibiotic.
o The MIC is found in the last tube where there is no visible growth of bacteria

This is a quantitative method that allows adjustment of dosage regimen in a particular patient. The MIC is
recommended to use when giving antibiotics to reduce the chance of the bacteria developing resistance,
then reproducing and creating more resistant copies.
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The serial dilution method is very time consuming, so usually an E test is performed, which does not find the
exact MIC but it still good enough for routine work.

- A petri dish is prepared with a strip of paper containing known concentrations of antibiotics. They
are left to cultivate at 37C for 24hrs
- The MIC is seen at the section of the strip where the bacteria are just starting to grow, as shown:

- This method allows for testing of fastidious and anaerobic bacteria

17. Antibiotics and chemotherapeutics; characteristics and usage

Characteristics

Antibiotics should have a wide spectrum of activity with the ability to destroy or inhibit the different species
of pathogenic microorganism; it should be non-toxic to the patient and produce no side effects, allergic
reactions, and should not eliminate the natural flora of the patient.

- Summarise points 16 and 17 from part 1

18. Disk Diffusion Method for Antimicrobial Susceptibility Testing – antibiogram

(NCCLS method)
Disk diffusion method

This is a qualitative method that allows for categorisation of bacteria from a patient into 3 groups: sensitive
(S), intermediate (I) or resistant (R), depending on it reacts to an antibiotic. It can also be done to decide if
combination therapy will be effective, or to find the genetic mechanism underlying a bacteria’s resistance.

Principle

Filter papers containing known concentrations of antibiotic are placed onto a petri dish that has been
inoculated with the organism of interest. This will be done to determine how well the antibiotic can
penetrate into the agar

- Because the disk is circular in shape, the antibiotic will penetrate in all directions at equal velocities.
Microbiology – Practical syllabus

- The highest concentration of antibiotic is seen beneath the filter paper; the concentration then
diminishes as the antibiotic travels further into the periphery.
- No growth occurs on the areas of the petri dish where the concentration of drug is high enough to
cause inhibition of growth, creating a “zone of inhibition” around the disk.

Method

A pure culture from a patient is needed for this method. A sterile cotton tip is used to transfer a sample from
the pure culture into a saline-filled tube.

- The amount of bacteria transferred to the saline solution should be such an amount that creates a
solution that is standardised to 0.5 MacFarland units.
- When the MacFarland standard is achieved, a new sterile cotton tip is used to take a sample from
the newly standardised saline-bacteria solution and transfer it to a new petri dish.
o A sample should only be taken from the solution once
- The new petri dish is usually that of a Mueller-Hinton agar, or blood agar for bacteria with a low
metabolic activity.
- The bacteria sample is spread over the petri dish in 3 directions, then spread over the periphery to
ensure as much area is covered by the bacteria as possible.
o A maximum of 6 disks containing antibiotics of known concentrations are placed
symmetrically and equidistant from each other on the petri dish. The diameter of each disk
is always 6mm
o The disk is then left to cultivate at 37C for 24hrs

Pictured on the right is the expected result after 1 day of cultivation:

The diameter of the zone of inhibition of each disk is measured and an NCCLS
table is used to determine the sensitivity of the bacteria to each specific
antibiotic, which are categorised into S, I or R.

- NCCLS = national committee for clinical and laboratory standards

- Only antibiotics that are determined to be “sensitive” should be used.
o An “intermediate” result indicates that some bacteria have resistance, so if an intermediate
antibiotic is chosen, it should be administered in combination or at a higher concentration
within a shorter period of time. This may produce negative side effects.
- If two zones of inhibition fuse, this means that both antibiotics are more effective when
administered in combination
- If a zone suddenly cuts off, the bacteria may show some resistance.

19. Methods of sterilization. Usage of the particular methods in medical practice.

Methods of sterilization

Sterilization is defined as the process of killing all microorganisms, including bacterial spores, using heat,
chemical agents, filtration, irradiation, or a combination of methods. Sterilisation is always needed to kill
prions and spores. Disinfection does not kill these organisms.

Before disinfection or sterilisation, instruments need to be properly cleaned

Physical methods  can be categorised into 5 groups:

Microbiology – Practical syllabus

- Sunlight  common method that does not kill spores

- Drying  uncommon
- Dry heat  the sterilising mechanism behind dry heat is basically the oxidation of cell components.
A common example is the use of a Bunsen burner to sterilise a metal loop
o A hot air oven uses dry heat to sterilise glassware at 160C for 2 hours/180C for 1 hour.
- Moist heat  methods are categorised into below 100C, at 100C and above 100C.
o At 100C  e.g. boiling; good for clearing vegetative forms. Sterilises an instrument when
repeated 3x.
o Above 100C  e.g. autoclave
- Radiation  ionising or non-ionising

Heat

An autoclave uses saturated steam under pressure for sterilisation; works at 121C, 1-1.5atm for 15-20 mins.
Inside the autoclave is a metal box containing the instruments to be sterilised. The box is closed, then the
autoclave is filled with water and sealed closed.

- The temperature is gradually increased from 0 to 121C

- At 100C, the water begins to boil but vapour does not escape the system because it has been sealed
o The pressure from the vapour causes the pressure within the autoclave to increase; this kills
any microorganisms inside the metal box
o Afterwards, the instruments are dried at room temperature and then packaged into sterile
envelopes for transport
- The destruction of enzymes and membranes is influenced by the availability of water that disrupts
hydrogen bonds
- Steam under pressure aids penetration of heat into the instruments to be sterilised
- The usual cycle of 121C at 1atm is enough to kill spores of Clostridium botulinum, but spores of
some soil organisms are able to withstand this temperature.
o Prions are very resistant and need extra care to be sterilised
o When instruments are dried at room temperature, they may be contaminated again by the
open air. Additionally, they may be contaminated by the packaging.
o Disinfectants used on the instruments may remain on the instruments for a while and will
need to be washed off. The water used to wash them may not be sterile
o For the above reasons, it is necessary to re-sterilise the instruments using a hot air oven.

A hot air oven uses dry heat at 160-180C for 1-2 hours; oxidative destruction of cells. A Bunsen burner is
another example of use of dry heat to cause oxidative damage to cells.

Tyndallisation  designed to reduce the level of activity of sporulating bacteria that may remain after a
simple boiling water method

- Boil for 20 minutes at atmospheric pressure, then cool, incubate for a day and repeat 3x
- The 3 incubation periods allow heat-resistant spores to germinate and form the heat-sensitive
vegetative state, which are then killed by the next boiling step

Filtration

Modern filters made of nitrocellulose work by electrostatic attraction and physical pore size to retain
organisms. The result is a particle-free fluid

- Does not sterilise objects

Microbiology – Practical syllabus

- Generally removes most bacteria and viruses

- Can be done under negative or positive pressure

Chemical methods

Used to sterilise heat-sensitive materials such as plastics

Gases  ethylene oxide and formaldehyde are both alkylating gases that kill microorganisms by damaging
proteins and nucleic acids

- Ethylene oxide is not usually used because it is toxic and potentially explosive
- Formaldehyde is not explosive but it is an irritant to mucous membranes. It is used as a disinfectant

Liquid  glutaraldehyde is less toxic than formaldehyde; used for disinfection but does not sterilise

- Instruments are immersed in solution for about 20 minutes. In case of spores, they are immersed for
2-3 hours.

Irradiation

Ionising  UV and infrared; UV is inefficient as a sterilant, but it is used in hospital settings to inhibit the
growth of bacteria in air such as in operating room and TV laboratories.

- UV has poor penetration in most materials hence it is inefficient

- Cannot be used on humans because it alters DNA structure

Non-ionising  x-rays and gamma-rays; gamma is used to sterilise large batches of small instruments such
as needles, syringes, IV lines, gloves etc.

- The mechanism involves production of free radicals which break bonds in DNA.
- Irradiation kills spores at a higher dose than vegetative cells because of the relative lack of water in
spores.

20. Disinfectants. Usage of the particular disinfectants in medical practice

Definition

Disinfection is defined as the killing or removing of harmful vegetative microorganisms. The difference
between sterilising and disinfecting is that sterilisation includes the killing of prions and spores, whereas
disinfection kills only harmful vegetative microorganisms.

- Sterilisation is always needed to kill prions and spores

- A high level of disinfection is needed to kill mycobacteria, non-enveloped viruses and fungi
o A high level of disinfection is not the same as sterilisation because it cannot kill spores
- A medium level is needed to kill vegetative bacteria and enveloped viruses.

Disinfection only uses chemical methods to remove microorganisms, whereas sterilisation uses various
methods. The activity of disinfectants is directly proportional to temperature and concentration (up to a
point called the optimal concentration; after this level there is no effect in increasing concentration further).
Disinfectants may be inactivated by:

- Dirt, organic matter, plastics

- Proteins, pus, blood, mucus, faeces
Microbiology – Practical syllabus

o These materials need to be cleaned off first before disinfectants

Halogens

Iodine compounds are the most effective halogen used for disinfection; they work by precipitating proteins
and oxidising essential enzymes. A disadvantage of iodine is that it stains and is irritating

- An iodophor is a combination of iodine and an organic molecule; they do not stain and are less
irritating than iodine. E.g. isodine, betadine.

Chloride gas  used for disinfecting water supplies, swimming pools, sewage etc. ordinary household bleach
is composed of sodium hypochlorite.

- Mix with water to produce a strong oxidant called hypochlorous acid, which is uncharged and enters
cells easily.
- Chloramines  derived from ammonia by substitution of H+ with Cl-. These are used for disinfection
of water (dissolvable tables).
- Some disadvantages of using chloride compounds as disinfectants are:
o Some irritate the skin, nose, eyes
o May not completely eliminate certain organisms from contaminated water, such as the
fungus Cryptosporidium (water should be boiled instead).

Fluorine has antimicrobial properties that contribute to the prevention of dental caries (cavities). Fluoride is
the active ingredient of toothpaste and is also commonly added to tap water to help communities maintain
oral health.

- Chemically, fluoride can become incorporated into the hydroxyapatite of tooth enamel, making it
more resistant to corrosive acids produced by the fermentation of oral microbes.
- Enhances the uptake of calcium and phosphate ions in tooth enamel, promoting remineralization.
- Bacteriostatic  fluorine accumulates in plaque-forming bacteria, interfering with their metabolism
and reducing their production of the acids that contribute to tooth decay.

Others

Phenolics  tend to be stable, persistent on surfaces, and less toxic than phenol. They inhibit microbial
growth by denaturing proteins and disrupting membranes.

- Triclosan  a bisphenol compound commonly used in hand soaps and frequently incorporated into
products such as cutting boards, knives, shower curtains, clothing, and concrete, to make them
antimicrobial.
o It is particularly effective against gram-positive bacteria on the skin, as well as certain gram-
negative bacteria and yeasts

Alcohols  work by rapidly denaturing proteins, which inhibits cell metabolism, and by disrupting
membranes, which leads to cell lysis. Once denatured, the proteins may potentially refold if enough water is
present in the solution.

- Alcohols are typically used at concentrations of about 70% aqueous solution and work better in
aqueous solutions than 100% alcohol solutions. This is because alcohols coagulate proteins.
o In higher alcohol concentrations, rapid coagulation of surface proteins prevents effective
penetration of cells.
Microbiology – Practical syllabus

- The most commonly used alcohols for disinfection are ethanol and isopropyl alcohol (isopropanol;
rubbing alcohol).

21. Serological diagnosis; principles, techniques, and application of diagnosis of

infectious disease.
Principles

Serological techniques that are used to diagnose disease are based on the antigen-antibody reaction; i.e.,
the binding of an antigen with its specific antibody, which is best detectable when each are at optimal
concentrations.

There are two types of serological reaction: direct and indirect:

- Direct  used for identification of an unknown organism, detection of microbial antigens by specific
(known) antibodies, and for serogrouping/serotyping of isolated organisms.
o Examples  slide agglutination (point 22), tube agglutination (point 23)
- Indirect  detection of specific and non-specific Abs (IgM and IgG) using antigens or organisms.
o Examples  see point 24

22. Slide agglutination (direct agglutination): principle, technique, and reading of

the results.
Principle

Agglutination is a specific antigen-antibody reaction where the antigen is cell-associated; the positive
reaction is observed as an aggregation or clumping if the agglutinogen is homologous to the antibody. With
this method, several agglutinations can be performed on the same slide.

There is a correlation between the serotype and pathogenic property of E. coli sera, corresponding to
enteropathogenic E. coli

Technique

- Place a drop of serum onto a clean glass slide

- Use a sterile loop to take a sample of a 24hr pure agar culture and suspend it into the first drop of
the serum; mix well
- Sterilise the loop and repeat the process with a 2nd drop of serum and subsequent sera.
- Observe the slide with your naked eye against a black background and preferable over a concave
mirror; determine the EPEC sera
o The bacteria most likely belong to the serotype used in the preparation of the corresponding
serum.

Any agglutination that occurs 5 seconds after suspension of the culture in the serum should be disregarded
as delayed agglutination and does not have any diagnostic value.

EPEC is responsible for diarrhoea and enterocolitis in children under 3 years.

Positive result  agglutination.

Microbiology – Practical syllabus

23. Widal’s serodiagnosis (tube agglutination method) using a patient’s serum and
Salmonella antigens 0:9,12 and H:2. Principle, technique, and reading of the results.
Principle

Agglutination tests are widely used in underdeveloped countries that may lack appropriate facilities for
culturing bacteria. For example, the Widal test, used for the diagnosis of typhoid fever, looks for
agglutination of Salmonella enterica subspecies typhi in patient sera.

The Widal test is rapid, inexpensive, and useful for monitoring the extent of an outbreak; however, it is not
as accurate as tests that involve culturing of the bacteria.

- Allows for the detection of specific agglutinins corresponding to the known antigen

Technique

- Prepare a 1/50 dilution of patient serum and distribute into a series of tubes
o 0.5mL saline in all tubes except for the first one of the O range
 Take 0.5mL of the diluted serum from the 1st and 2nd tubes of the O range and put it
into the first tube of the H range.
o Take 0.5mL of the mixture from the 2nd tube of O range and put it into 3rd O range tube
o Take 0.5mL from 3rd O range tube and put it into 4th O range tube.
 Take 0.5mL from 4th O range tube and discard it
o Use tube 5 as a control
o Serial dilute H range tubes in the same way as the O range tubes, using the 5th tube as a
control.
- add 2 drops of antigen O:9,12 to all tubes of the O range
- add 2 drops of antigen H:d to all tubes of the H range.
- Incubate at 37C for 2 hours, after which they are left at room temperature

Result

Titre higher than 1/100 for O agglutination and higher than 1/200 for H agglutination are positive for typhoid
fever.

The Widal test frequently produces false positives in patients with previous infections with other subspecies
of Salmonella, as well as false negatives in patients with hyperproteinemia or immune deficiencies. A culture
should be performed if possible for confirmation.

24. Passive haemagglutination, co-agglutination, latex agglutination. Principle,

technique, and results.
Passive haemagglutination

Principle  RBCs are used to adsorb a soluble antigen onto their surfaces; the RBCs then agglutinate in the
presence of antiserum specific for the adsorbed antigen. These antigens can be carbohydrate or protein
structures.
Microbiology – Practical syllabus

- The technique is called “indirect” or “passive” haemagglutination because it is not the antigen of the
erythrocytes that are bound by antibody, but those that are passively attached to the cells.
- E.g., in rubella antibody tests, erythrocytes are coated with rubella antigen.
- Erythrocytes from sheep or rabbits, or from blood type O humans are used.

Result  in presence of antibody, agglutination occurs (smooth mat forms). In absence (i.e., a negative
result), sedimentation of erythrocytes is seen (compact button of cells forms).

Co-agglutination

Staphylococcus aureus is used to aid indirect agglutination; protein A on S. aureus can bind the Fc region of
immunoglobulins, then the Fab regions of these Igs can bind an antigen to be examined for serology testing.

- The antibodies retain their antigen recognition properties

The IgG-coated S. aureus cells are then mixed with the specimen (e.g., patient serum) or an antigen
preparation to detect the presence of specific antigens.

- A positive result is agglutination of the cells; this demonstrates the presence of the specific antigen
in the sample or preparation.

Latex agglutination

An indirect agglutination assay in which antibodies are identified in a patient’s serum using specific antigens
that are attached to latex beads.

- When mixed with patient serum, the antibodies will bind the antigen, cross-linking the latex beads
and causing the beads to agglutinate indirectly; this indicates the presence of the antibody.
- This technique is most often used when looking for IgM antibodies, because their structure provides
maximum cross-linking.
- One example of this assay is a test for rheumatoid factor (RF) to confirm a diagnosis of rheumatoid
arthritis. RF will agglutinate IgG-coated latex beads.

In the reverse test, soluble antigens can be detected in a patient’s serum by attaching specific antibodies
(commonly mAbs) to the latex beads and mixing this complex with the serum.

Latex agglutination uses polystyrene beads coated with specific antibodies.

Positive result  agglutination; indicates the presence of the antigen/antibody of interest.

25. Ring precipitation test (thermoprecipitation). Principle, technique, and results.

Principle

Also called thermo-precipitation of Ascoli or ring precipitation of Ascoli; used when infection with Bacillus
anthracis is suspected. Tissue is taken from these individuals and high temperature is used to extract
antigens of Bacillus anthracis. i.e., used for the diagnosis of anthrax

Technique

Reagents used in this reaction are:

- Specific Ig against B. anthracis antigens (Bacillus anthracis produces a heat-stable antigen).

- Thermo-extract from a cadaver that died of the infection (antigens of B. anthracis)
Microbiology – Practical syllabus

- Thermo-extract from a living individual that is ill with infection with B. anthracis (antigens)
- Thermo-extract from a pure culture of B. anthracis (antigens)
- Thermo-extract from a healthy living individual (no antigen)
- Saline
- Non-specific Igs that cannot react with B. anthracis antigen.

6 tubes are used. 0.2mL of specific Ig is added to all tubes except the last.

- Thermo-extract from different sources are added to specific tubes, so that each tube has 2 reagents
each and contain 0.4mL of sample in total.

The tubes are prepared as follows:

Reagent Tube
1 2 3 4 5 6
Ig against B. anthracis antigen 0.2 mL 0.2 mL 0.2 0.2 mL 0.2 mL -
mL
Extract from a cadaver (Ag of B. anthracis) 0.2 mL
Extract from an ill person (Ag of B. anthracis) 0.2 mL
Thermo-extract from a pure culture (Ag) 0.2 0.2 mL
mL
Thermo-extract from healthy individual (no Ag) 0.2 mL
Saline 0.2 mL
Non-specific Igs that cannot react with Ag 0.2 mL

Results

A positive reaction is expected in tubes 1-3, which contain the B. anthracis antigen, and a negative result in
4-6 because these tubes do not have the antigen.

- Positive  development of a white ring at the junction of antiserum and antigen solution (the serum
has a higher density and falls on the bottom of the tube).
- Negative  absence of ring formation.

26. Ouchterlony double immunodiffusion (DID). Radial single immunodiffusion.

Immuno-electrophoresis. Principle, application, results.
Ouchterlony double immunodiffusion

Ouchterlony reaction  used for qualitative determination of Ag; to see if different Ags have the same
structure, or different, or similar

- A gel with a well in the middle is prepared. Into the well is inserted a known Ig.
- Different antigens are placed in 2 more wells that surround the middle well.
- Precipitation lines will form if specificity exists between the components (i.e., the Ag and Ab).
o If 2 precipitation lines fuse, this means the 2 antigens are identical in structure.
o If 2 lines cross, this means the 2 antigens are completely different in structure
o If 2 lines are semi-crossed and semi-fused, this means the 2 antigens are similar in structure
but not identical.
Microbiology – Practical syllabus

Radial single immunodiffusion

Also called Macini method  used for quantification of Ag; the Ag that would be quantified is an
immunoglobulin.

Principle: Igs are glycoproteins. The strongest stimulators for the immune system are proteins and sugars,
hence Igs can act as antigens (especially if serum Ig from one person is introduced into the system of another
person).

- A gel is prepared containing a known-Ig that will react with an unknown Ag (in patient serum).
o A visible reaction demonstrates that the Ig that is inoculated into the gel is specific to the Ag
in the patient’s serum.
o The concentration of the sera (both control and patient) decreases from high to low in
different wells in the gel.
- Antigen then diffuses radially from the well and a precipitin ring forms at the point of antibody-
antigen equilibrium. Antigen concentrations are determined by measuring the diameter of precipitin
rings and extrapolating using standard curves.

A graph can be used to demonstrate if the concentration of the patient’s Ig Ag is within normal or abnormal
parameters when compared against the control Ag Ig.

- E.g., a higher IgE in patient sera is indicative of allergy or parasitic infection.

Immuno-electrophoresis

This method combines electrophoresis in agar and gel and immunodiffusion; it is used for qualitative analysis
of antigens. The antigens are electrophoresed and separated based on their molecular weight.

- Wells are cut into a gel

- Antigens are placed into the wells and a charge is applied to separate the components of the antigen
mixture according to size
- Antiserum containing immunoglobulins is added to the opposite end of the gel and moves towards
the antigen components, resulting in the formation of separate precipitin lines in 18-24 hours.
o Each line indicates a reaction between individual proteins with its antibody.

Results  the presence of elliptical precipitin arcs represents antigen-antibody interaction. The absence of
the formation of precipitate suggests no reaction.

- Different antigens (proteins) can be identified based on the intensity, shape, and position of the
precipitation lines.

PCR uses electrophoresis for detecting multiplied DNA/RNA; they cannot be visualised with the naked eye so
a substance called ethidium bromide is added into the gel. It has the property to bind to DNA/RNA
molecules

- The gel is then exposed to UV and the DNA/RNA molecules start to glow.
- The samples are compared against a control of known base pairs
Microbiology – Practical syllabus

27. Titration of haemolytic serum; application, principle, and results

Principle

This uses the concept that IgG or IgM activate complement onto the RBCs, leading to MAC formation and cell
lysis (haemolysis) when Igs bind to surface antigens on erythrocytes. The formation of MAC due to
immunoglobulin binding occurs in the classic complement pathway.

To prepare we need:

- Serum from patient containing Ig (in decreasing concentration with each successive tube)
o No serum is added to the last tube to use as a control; saline is added instead
- 2% sheep blood cells in all tubes; Sheep blood is very similar to human blood
- Complement in all tubes
- It is left for 1 hour at 37C
o Eventually the concentration of Ig will be too low for haemolysis of all cells in a tube to
occur, so the erythrocytes will collect in a pellet at the bottom of the tube
o The lowest concentration that will visibly produce haemolysis corresponds to the “titre”

Full haemolysis in the first tube but none in the last tube

28. Complement fixation reaction for diagnosis of syphilis: Wassermann’s reaction.

Application, principle, and results.
Complement fixation test

Syphilis is caused by Treponema pallidum, which cannot be stained by gram staining. It is tested for by a
serology reaction called Wasserman. This reaction uses:

1. One system of reagents containing:

a. Cardiolipin antigens that are similar to the antigen of T. pallidum. These are used to detect
Igs within the patient’s serum, which will be present if the patient is ill with syphilis infection.
If they are healthy, there will be no Ig in their serum against T. pallidum.
Microbiology – Practical syllabus

b. Serum from patient

c. Complement
d. 1hour at 37C
2. A second system of reagents containing:
a. Erythrocytes
b. Ig against the erythrocytes
c. 1hour 37C

Both systems are added to the same tube; the first one is added, then after 1 hr the second system is added.

- A sick patient’s serum will contain antibodies that can bind cardiolipin antigen; this is due to the
similarity between the cardiolipin antigen and the T. pallidum antigen.
o Complement will be activated against the bound antibody.
o When the 2nd reagents are added, the complement proteins will not bind to the Igs against
the erythrocytes because they are already occupied by the antibodies that are bound to
cardiolipin antigen.
o Therefore, haemolysis will not occur in this sample.
- A healthy patient’s serum will not contain antibodies that can bind cardiolipin antigen, because they
have not been exposed to T. pallidum antigen.
o When the 2nd reagents are added, the complement system will be activated by the
immunoglobulins that bind the erythrocytes
o Therefore, haemolysis will occur in this sample

Hence a positive result (demonstrating that this person is infected with syphilis) is the absence of haemolysis
in a tube.

29. ELISA (enzyme-linked immunosorbent assay); basic principle, reading, and

interpretation of results, application. Immunofluorescence; direct and indirect
immunofluorescence, application.
ELISA

There are 3 types of ELISA; direct, indirect, and sandwich.

Direct  antigens are immobilized in the well of a microtiter plate. An enzyme-conjugated antibody that is
specific for a particular antigen is added to each well. If the antigen is present, then the antibody will bind.
Microbiology – Practical syllabus

- After washing to remove any unbound antibodies, a colourless substrate (chromogen) is added. The
presence of the enzyme converts the substrate into a coloured end product.
- While this technique is faster because it only requires the use of one antibody, it has the
disadvantage that the signal from a direct ELISA is lower (lower sensitivity).

Indirect  used to detect an antibody against a specific pathogen; the indirect ELISA starts with attaching
known antigen (e.g., peptides from HIV) to the bottom of the microtiter plate wells.

- After blocking the unbound sites on the plate, patient serum is added; if antibodies are present
(primary antibody), they will bind the antigen.
- After washing away any unbound proteins, the secondary antibody with its conjugated enzyme is
directed against the primary antibody (e.g., antihuman immunoglobulin).
o The secondary Ab allows us to quantify the antigen-specific Ab present in the patient’s
serum by the intensity of the colour produced from the enzyme-chromogen reaction.

Sandwich  to detect antigen; The first step is to add the primary Ab to the wells of a microtiter plate. The
Ab sticks to the plastic by hydrophobic interactions. After an incubation time, unbound Abs are washed off.

- A blocking protein is then added (e.g., albumin) to bind the remaining nonspecific protein-binding
sites in the well.
- The antigen is added next; The primary antibody captures the antigen.
- Following a wash, the secondary antibody is added, which is conjugated to an enzyme. After a final
wash, a chromogen is added, and the enzyme converts it into a coloured end product.
o The colour intensity caused by the end product is measured with a spectrophotometer.
o The amount of colour produced (measured as absorbance) is directly proportional to the
amount of enzyme, which in turn is directly proportional to the captured antigen.

Immunofluorescence

IF methods can be utilised for the detection of specific microbial antigens and antibodies; direct or indirect
approaches can be used.

- Direct  a labelled mAb binds an antigen

- Indirect  a secondary polyclonal antibody binds patient antibodies that react to a prepared antigen

Fluorescently labelled antibodies can be used to quantify cells of a specific type using flow cytometry, which
is a cell-counting system that detects fluorescing cells as they pass through a narrow tube one cell at a time.
E.g., in HIV infection, it is important to know CD4 T cell counts in the patient’s blood;

- analysis begins by incubating a mixed-cell population (e.g., WBCs from a donor) with a fluorescently
labelled mAb specific for a subpopulation of cells (e.g., anti-CD4).
- The cells are then introduced to the flow cytometer
through a narrow capillary that forces the cells to pass
in single file. A laser is used to activate the fluorogen.
- The fluorescent light radiates in all directions, so the
detector can be positioned at an angle from the light.
o Data is collected from the forward- and side-
scatter detectors
o Forward scatter indicates cell size, side scatter
indicates cell complexity or granularity.
Microbiology – Practical syllabus

The results are compiled as a histogram that has 2 peaks; the peak with the lower fluorescence reading is
indicative of the cell population that is not bound by a fluorogen, so they are not bound to antibody and do
not express CD4. The peak with the high reading represents cells that express CD4.

30. Antibacterial vaccines; characterisation, application | 31. Antiviral vaccines.

Vaccines

Vaccination is a form of artificial immunity. By artificially stimulating the adaptive immune defences, a
vaccine triggers memory cell production similar to that which would occur during a primary response.

This allows a patient to mount a strong secondary response upon exposure to the pathogen without having
to suffer through an initial infection.

Types of vaccines

Live attenuated vaccines  these expose an individual to a weakened strain of a pathogen by establishing a
subclinical infection that will activate the adaptive immune defences. Pathogens are attenuated to decrease
their virulence via methods such as genetic manipulation (to eliminate key virulence factors) or long-term
culturing in an unnatural host or environment (to promote mutations and decrease virulence).

- By establishing an active infection, live attenuated vaccines stimulate a more comprehensive

immune response than other vaccines; they can activate both cellular and humoral immunity and
stimulate the development of memory for long-lasting immunity.
- Disadvantages  potential for a patient to develop signs and symptoms of disease during the active
infection (in immunocompromised patients), and a risk of the attenuated pathogen reverting back to
full virulence.

Inactivated vaccines  contain whole pathogen that have been killed or inactivated with heat, chemicals, or
radiation. For inactivated vaccines to be effective, the inactivation process must not affect the structure of
key antigens on the pathogen.

- inactivated vaccines do not produce an active infection, and the resulting immune response is
weaker and less comprehensive than that provoked by a live attenuated vaccine.
- Typically, the response involves only humoral immunity
- inactivated vaccines usually require higher doses and multiple boosters, possibly causing
inflammatory reactions at the site of injection.
- Also, there is no risk of causing severe active infections, but some side effects may still manifest.

Subunit vaccines  exposes the patient only to the key antigens of a pathogen, not whole cells or viruses.
Subunit vaccines can be produced either by chemically degrading a pathogen and isolating its key antigens or
by producing the antigens through genetic engineering.

- These vaccines contain only the essential antigens of a pathogen, so the risk of side effects is low.

Toxoid vaccines  also do not introduce a whole pathogen to the patient; they contain inactivated bacterial
toxins, called toxoids. Toxoid vaccines are used to prevent diseases in which bacterial toxins play an
important role in pathogenesis. These vaccines activate humoral immunity that neutralizes the toxins.

Conjugate Vaccines  a type of subunit vaccine that consists of a protein conjugated to a capsule
polysaccharide. Conjugate vaccines were developed to enhance the efficacy of subunit vaccines against
pathogens that have polysaccharide capsules that help them evade phagocytosis.
Microbiology – Practical syllabus

- Subunit vaccines against these pathogens introduce T-independent capsular polysaccharide antigens
that result in the production of antibodies that can opsonize the capsule and combat the infection;
- The conjugated protein-polysaccharide antigen stimulates production of antibodies against both the
protein and the capsule polysaccharide.

32. Immune sera; characterisation, application

Characterisation

Immune sera are biological preparations containing antibodies against a microorganism or its exotoxin,
which are applied to provide specific passive immunity against it.

- Antibodies are produced by plasma cells; when an antigen binds to the B-cell surface, it stimulates
the B cell to divide and mature into plasma cells.
- The first antibody made after exposure to an antigen is IgM.

Immune sera can be acquired from an animal (horses, pigs) that has been immunised either by antigen
infection or infection with the microorganism containing the antigen.

Application

Artificial passive immunity refers to the transfer of antibodies produced by a donor (human or animal) to
another individual. This transfer of antibodies may be done as a prophylactic measure (i.e., to prevent
disease after exposure to a pathogen) or as a strategy for treating an active infection.

For example, artificial passive immunity is commonly used for post-exposure prophylaxis against rabies,
hepatitis A, hepatitis B, and chickenpox (in high-risk individuals).

- Active infections treated by artificial passive immunity include CMV infections in

immunocompromised patients and Ebola virus infections.
- Artificial passive immunity is also used for the treatment of diseases caused by bacterial toxins,
including tetanus, botulism, and diphtheria.

In contrast, natural passive immunity refers to the natural passage of antibodies from a mother to her child
before and after birth, either via the placenta (IgG) or via breast milk (IgA).

33. Immunomodulators (immunostimulators); characterisation, application

Characterisation

Immunostimulators are substances (drugs and nutrients) that stimulate the immune system by inducing
activation or increasing activity of any of its components.

There are two main categories of immunostimulators:

- Specific immunostimulators  provide antigenic specificity in immune response, such as vaccines or

any antigen (describe types of vaccines from point 30-31).
- Non-specific immunostimulators  act irrespective of antigenic specificity to augment immune
response of other antigen or stimulate components of the immune system without antigenic
specificity, such as adjuvants and non-specific immunostimulators.
Microbiology – Practical syllabus

Many endogenous substances are non-specific immunostimulators. For example, female sex hormones are
known to stimulate both adaptive and innate immune responses. Other hormones appear to regulate the
immune system as well, such as prolactin, growth hormone and vitamin D.

Application

Stimulates the immune system.