By
Dr. Emad AbdElhameed Morad
Lecturer of Medical Microbiology and Immunology

 Microscopic examination.
 Cultural appearance.
 Biochemical reactions.
 Serological identification.
 Animal inoculation.
 Bacteriophage typing.
 Molecular methods.

 Examination of wet mount for motility.
 Examination of Gram stained preparation to demonstrate:
1) Size
2) Shape
3) arrangement
4) Staining affinity

 Pigment production: o Endopigment (restricted to the colonies):
• Golden yellow with Staphylococcus aureus.
• Lemon yellow with Staph. saprophyticus.
• White with Staph. epidermidis.
o Exopigment (the color diffuses in the surrounding medium):
• Green exopigment with Pseudomonas aeruginosa.

 Hemolysis on blood agar: o Complete (beta) hemolysis:
• Staphylococcus aureus and Streptococcus pyogenes .
o Partial (alpha) hemolysis:
• Streptococcus viridans and pneumococci.
o No (gamma) hemolysis:
• Enterococci.

 Effect on lactose of MacConkey’s agar: o Lactose fermenters:
• Appear as rose pink colonies.
• Example: E. coli & klebsiella.
o Non Lactose fermenters:
• Appear as pale colonies.
• Example: salmonella & shigella.

1) Sugar fermentation: o Composed of:
Six tubes.
Each tube contains peptone water + specific sugar in a concentration of 1%.
The sugars are glucose, lactose, maltose, mannite, sucrose, salicin.
Each tube contains also Andrade’s indicator which changes to pink if acid is produced.
An inverted tube (Durham’s tube) is placed in the medium.
Gas production is revealed by collection of bubbles at its apex.

o Used to:
Differentiate between bacteria according to their fermentative effect on different sugars and whether the fermentation is associated with gas production or not.
Fermentation of glucose, maltose, mannite with acid only
Fermentation of all sugars with acid + gas production

2) Indole test o Principle:
Demonstrates the ability of certain bacteria to decompose the amino acid tryptophan present in peptone water to indole.
Indole is then tested for by adding few drops of Kovac’s reagent which gives a pink ring in the presence of indole.
o Procedure:
The organism is inoculated in peptone water and after incubation at 37 degree for 24 hours, Kovac’s reagent is added. o Interpretation:
If a pink ring is produced, the organism is indole +Ve (E. coli).
If a yellow ring is produced, the organsim is indole –Ve
(Klebsiella).

3) Voges-Proskauer’s reaction (VP): o Principle:
Some bacteria ferment glucose with production of acetyl methyl carbinol.
o Procedure:
Bacteria is grown in glucose phosphate peptone water for 48 hours.
Then KOH is added to test for acetyl methyl carbinol formation.
o Interpretation:
If an eosin pink color is produced, VP reaction is +Ve.

4) Methyl red test (MR): o Principle:
Detects the ability of some bacteria to produce large amounts of acid on fermentation of glucose, thus lowering the pH of the medium below 4. o Procedure:
The bacteria is grown in glucose phosphate peptone water.
After incubation at 37 degree for 48 hours, few drops of methyl red indicator are added.
o Interpretation:
A positive test gives bright red color.
A negative test gives a yellow color.

5) Urease test: o Principle:
Some organisms produce urease enzyme.
This enzyme splits urea with the release of ammonia.
Ammonia causes alkalinity and increases pH of the medium.
The phenol red indicator turns deep pink.
o Procedure:
The bacteria is grown on a medium containing urea and phenol red indicator for 24 hours.
o Interpretation:
Urease positive organisms such as proteus will turn the medium deep pink.

+Ve -Ve

6) Oxidase test: o Principle:
Some bacteria produce oxidase enzyme which reduces the oxidase reagent (tetramethyl-p-phenylene-diamine hydrochloride) to a deep purple color.
o Procedure:
Done by picking up a portion of the colony tested and smearing it on a filter paper impregnated with oxidase reagent.
o Interpretation:
The immediate development of a deep purple color indicates positive test.
Examples: neisseria & pseudomonas & vibrios.

7) H2S test: o Principle:
Production of H2S from proteins.
o Procedure:
Inoculate the organism on peptone water.
After incubation, put lead acetate paper at the mouth of the tube.
o Interpretation:
If the organism produces H2S, a black color is formed.
Examples: proteus & Salmonella typhi.

8) Citrate utilization test: o Principle:
Tests the ability of bacteria to utilize citrate as the only source of carbon as they have citrase enzyme that degrades citrate.
o Procedure:
The bacteria is grown on citrate agar slant which contains sodium citrate as the only source of carbon, ammonium phosphate as the source of nitrogen and bromothymol blue as pH indicator.
If the bacteria could use citrate as the only source of carbon, it will grow and convert ammonium phosphate to ammonia and ammonium hydroxide. Both molecules will turn the agar alkaline.
o Interpretation:
Blue color with streaks of growth on the agar means +Ve test such as klebsiella .
Green color with no growth means –Ve test such as E. coli.

9) Catalase test: o Principle:
Tests the ability of some bacteria to produce catalase enzyme.
o Procedure:
Pick up the test colony on a platinum loop and immerse it in few drops of 3 % H2O2 (hydrogen peroxide).
o Interpretation:
Rapid effervescence indicates oxygen production and a positive test.
This test is used to differentiate between staphylococci (+Ve) and streptococci (-Ve).
Examples of catalase positive bacteria are staphylococci, campylobacter and brucella.

Catalase +Ve

10) Coagulase test:
Slide method o Detects the coagulase bound to the organism.
o Homogenous suspension of the test organism is made in a drop of saline on a slide then mixed with a drop of undiluted human or rabbit plasma.
o Staphylococcus aureus clumps within 15 sec. because coagulase enzyme precipitates the fibrin in the plasma on the cell surface.

Tube method o Detects the free coagulase.
o It is done by adding 5 drops of an overnight broth culture of the test organism to 0.5 ml of human or rabbit plasma diluted
1/10 in sterile saline.
o The tubes are incubated for 6-12 hours and inspected hourly for coagulation.
o Staphylococcus aureus is coagulase positive.
Positive
Negative

11) DNAase test: o Principle:
This test is used to identify Staph. aureus as it produces
DNAase enzyme.
o Procedure:
The tested organism is cultured on a medium which contains
DNA.
After overnight incubation, the colonies are tested for DNAase production by flooding the plate with weak HCL.
The acid precipitates unhydrolyzed DNA.
o Interpretation:
DNAase producing colonies (such as Staph. aureus) are surrounded by clear areas due to DNA hydrolysis.

DNAase +Ve

12) Analytical profile index (API): o Principle:
It is composed of a plastic strip with cupules containing dehydrated substances. Each cupule has a small hole at the top.
o Procedure:
A saline suspension of the test organism is dropped in the cupules.
The strip is covered with a lid and placed in a humidified plastic chamber and incubated at 37 degree for 24-48 hours. o Interpretation:
Biochemical profiles are determined by reading the color change and interpret according to the available charts.
These are then converted to numerical codes which will be read from a profile index to identify the bacteria.

API

13) Automated bacterial identification systems: o Principle:
Examples: Vitek system, microscan, phoenix.
These systems identify the organism and its antibiotic sensitivity by detecting color changes or turbidity in special plastic cards inoculated with the organism.
Such cards are composed of tiny wells that contain substrates for detection of biochemical reactions and antibiotic sensitivity.
Once the card has been inoculated and placed in the instrument, it will automatically perform all readings.
Results are available within 4-6 hours.

Vitek system
Vitek card

- When a bacterium is injected in an experimental animal, the latter will respond by formation of antibodies.
If these antibodies are mixed with the same bacterium, agglutination occurs.
Thus, unknown bacterium can be identified by demonstrating its reaction with one of several known antibodies.

- The use of laboratory animals (mice, guinea pigs, rabbits) is now limited due to the advancement in medical microbiological techniques.
But they could be used in:
1) For growing the organisms that do not grow on culture such as lepra bacilli .
2) Isolation of bacteria from specimens containing small number of this bacteria.

3) To determine the virulence factor of an organism. For example if injection of diphtheria in a guinea pig caused its death , this means that the organism is toxigenic .
4) Isolation of certain pathogenic bacteria from a specimen containing many bacteria. For example, isolation of pneumococci form sputum. When sputum samples are injected in white mice , the animal will die after 24 hours and pneumococci could be isolated form its heart blood.

Guinea pig Mouse

Bacteriophages are viruses which infect the bacterial cells and cause their lysis.
Phage typing is used in epidemiological studies to trace the source of infection as in food poisoning.
Different types of a certain bacteria are lysed by different phage groups.
If a phage is added to a plate inoculated with susceptible bacteria, a zone of lysis will appear around the phage drop.

Bacteriophage typing
Lysis occurred in six areas corresponding to phages no. 6, 47, 53,
42E, 54, 77

1) Nucleic acid probes: o Principle
The probe consists of sequence of single stranded
DNA or RNA that forms a covalently bonded hybrid with the specific complementary strand of the nucleic acid of the organism.
The probe is labeled either to radioactive substance as iodine 131 or enzyme .
o Steps of hybridization
1) The bacterial cells are lysed to liberate DNA.

2) The DNA is treated by alkali or heat to separate the two strands.
3) The DNA strands are attached to nitrocellulose membrane.
4) Labeled probe is then applied to the membrane.
5) If the probe is complementary to the test DNA, it will form strong bonds and remain on the membrane after washing.
6) The probe is now treated to allow visualization using:
 If the label is a radioactive isotope, exposing the membrane to X-ray film will reveal a dark spot.
 Enzyme label leads to color change after adding a suitable substrate.

Steps of hybridization
The upper lane shows +Ve hybridization, the lower lane is -Ve

2) Polymerase chain reaction (PCR): o Principle
- It is a process of amplification of a short sequence of DNA or RNA leading to accumulation of several copies of that short sequence. o Steps
1) DNA denaturation:
 By heating target DNA at 95 degree, the two strands of DNA will be separated.

2) Primer annealing:
 The temperature is lowered (40-60 degrees) and two short synthetic pieces of DNA termed primers are added. These primers are complementary to the two ends of the Opposite strands of the DNA to be amplified.
 Each primer anneals to its complementary sequence on the DNA strands.
3) Primer extension:
 Taq DNA polymerase starts to add nucleotides to both primers leading to formation of two complementary strands.

 At the end of the first cycle, there will be four strands of the target DNA which will serve as templates for synthesis of new strands in the next cycle, thus resulting in 8 strands and so on.
 So, one million copies can be produced in
20 cycles.

3) Plasmid profile analysis: o Principle
Plasmid profile analysis is based on the fact that similar bacterial strains often carry similar types and numbers of plasmids. o Steps
1) Plasmid DNA is isolated from the bacterial cells.
2) Plasmid DNA is then digested by restriction enzymes.
3) The resulting fragments are electrophoresed through agarose gel.
4) Identical plasmids will show the same size and number of bands on agarose gel.

Lanes 1 and 2 show two identical strains, lane 3,4,5 show three identical strains, lane 6 shows a strain with a different plasmid pattern.
Plasmid profile analysis