Examining specimens to detect isolate and identify pathogens:

1- Microscopy

2- Culture techniques

3- Biochemical reactions

4- Serological identification

5- Molecular biology techniques

6- Bacteriophage typing

7- Animal pathogenicity
 Staining methods……
 simple staining
 negative staining
 impregnation staining
 differential staining
 INDICATION
 Isolationof bacteria in pure culture.
 To demonstrate their properties.
 To obtain sufficient pure growth for
preparation of antigen & for other tests.
 To determine sensitivity to antibiotics.
 To estimate viable count.
 To maintain stock culture.
Use of substrates and sugars to identify pathogens:

a- Sugar fermentation:
Organisms ferment sugar with production of acid only
Organisms ferment sugar with production of acid and gas
Organisms do not ferment sugar

b- Production of indole:
Depends on production of indole from amino acid tryptophan
Indole is detected by addition of Kovac’s reagent
Appearance of red ring on the surface

e- H2S production:
Depends on production H2S from protein or polypeptides
Detection by using a strip of filter paper containing lead acetate
c- Methyl red reaction (MR):
Fermentation of glucose with production of huge amount of acid
Lowering pH is detected by methyl red indicator

d- Voges proskaur’s reaction (VP):

Production of acetyl methyl carbinol from glucose fermentation
Acetyl methyl carbinol is detected by addition KOH
Color of medium turns pink (positive)

e- Action on milk:
Fermentation of lactose with acid production
Red color if litmus indicator is added
f- Oxidase test:
Some bacteria produce Oxidase enzyme
Detection by adding few drops of colorless oxidase reagent
Colonies turn deep purple in color (positive)

g- Catalase test:
Some bacteria produce catalase enzyme
Addition of H2O2 lead to production of gas bubbles (O2 production)

h- Coagulase test:
Some bacteria produce coagulase enzyme
Coagulase enzyme converts fibrinogen to fibrin (plasma clot)
Detected by slide or test tube method

i- Urease test:
Some bacteria produce urease enzyme
Urease enzyme hydrolyze urea with production of NH3
Alklinity of media and change color of indicator from yellow to pink
A- Direct serological tests:
- Identification of unknown organism
- Detection of microbial antigens by using specific
known antibodies
- Serogrouping and serotyping of isolated organism

B- Indirect serological tests:

- Detection of specific and non specific antibodies
(IgM & IgG) by using antigens or organisms
A- Genetic probes (DNA or RNA probes):
Detection of a segment of DNA sequence (gene) in unknown
organism using a labeled probe

Probe: consists of specific short sequence of labeled single-

stranded DNA or RNA that form strong covalently
bonded hybrid with specific complementary strand of
nucleic acid .

B- Polymerase chain reaction (PCR):

Amplification of a short sequence of target DNA or RNA Then
It is detected by a labeled probe

C- Plasmid profile analysis:

Isolation of plasmids from bacteria and determination of their
size and number compared with standard strains by agars
gel electrophoresis
6.Bacteriophage
Viruses that parasitize bacteria are
called bacteriophage or phage.
Phage brings about lysis of
susceptible bacterial cells.
Phage typing is useful in
distinguishing stain among
salmonella & staphylococcus.
There is correlation between
bacteriophage type & epidemic
source.
* Animal pathogenicity test:
Animals commonly used are guinea pigs, rabbits, mice

* Importance of pathogenicity test:

- Differentiate pathogenic and non pathogenic

- Isolation organism in pure form

- To test ability of toxin production

- Evaluation of vaccines and antibiotics

 Identification of a bacterial isolate from a patient
provides guidance in the choice of an appropriate
antibiotic for treatment
 Many bacterial species are not uniformly susceptible to
a particular anti-bacterial compound
 This is particularly evident among the
Enterobacteriaceae, Staphylococcus spp., and
Pseudomonas spp.
 The wide variation in susceptibility and high frequencies
of drug resistance among strains in many bacterial
species necessitates the determination of levels of
resistance or susceptibility as a basis for the selection
of the proper antibiotic for chemotherapy
 Information about sensitivity pattern of
strain is useful for selecting choice of
drug.
 This may be useful as an epidemiological
marker in tracing hospital Infection.
 Ex. Staphylococcus aurous sensitivity to
mercury salt.
 In other cases sensitivity of bacteria to
agent help in identification of organism.
 Plasmid profile & bacitracin typing also
further help in identification of bacteria