Bacterial diagnosis

1. 3 laboratory Bacterial
diagnosis

2. Triple sugar–iron (TSI)
The TSI test can differentiate enteric organisms
based on their abilities to reduce sulfur and
ferment carbohydrates.
Enterobacteriaceae, which are all gram-negative
bacilli capable of fermenting glucose with the
production of acid, and to distinguish the
Enterobacteriaceae from other gram-negative
intestinal bacilli.

3. inoculate each test organism by using stab-and-streak inoculation.
Do not fully tighten screw cap.
Incubate for 18 to 24 hours at 37°C.
Examine the color of both the stab and slant of agar slant cultures.
determine the type of reaction that has taken place (acid, alkaline, or none) and
the carbohydrate that has been fermented (dextrose, lactose, and/or sucrose, all,
or none) in each culture.
The TSI agar medium also contains sodium thiosulfate, a substrate for
hydrogen sulfide (H2S) production, and ferrous sulfate for detection of this
colorless end product.
also determine whether or not each organism was capable of H2S production.

4. 1. Alkaline slant (red)
and acid stab (yellow)
with or without gas
production (breaks in the
agar).
Only glucose
fermentation has
occurred.
2. Acid slant (yellow) and
acid stab (yellow) with or
without gas production.
Lactose and/or
sucrose
fermentation has
occurred.
3. Alkaline slant (red)
and alkaline stab (red) or
no change (orange-red)
stab.
No carbohydrate
fermentation has
occurred.

8. Nitrate Reduction test
The reduction of nitrates by some aerobic and facultative
anaerobic microorganisms occurs in the absence of
molecular oxygen, an anaerobic process.
In these organisms anaerobic respiration is an oxidative
process whereby the cell uses inorganic substances such as
nitrates (NO3-) or sulfates (SO42-) to supply oxygen that
is subsequently utilized as a final hydrogen acceptor
during energy formation.

9. Reagents: Reagents
• Solution A (sulfanilic acid),
• Solution B (α-naphthylamine),
• zinc powder.
inoculate each test organism into Trypticase nitrate broth tube by means of a
loop inoculation.
Incubate all cultures for 24 to 48 hours at 37°C.
Add five drops of Solution A and then five drops of Solution B to all nitrate
broth cultures.
a red coloration may develops in each of the cultures.
Add a minute quantity of zinc to the cultures in which no red color developed.
observations each organism was capable of nitrate reduction. Identify the end
product (NO2- or NH3+/N2), if any, that is present.

11. Phage typing
These phages are used to identify different strains of bacteria within a
single species.
bacteriophages, may infect only a single strain of bacteria.
For outbreak infections.
Phage typing is a method used for detecting single strains of bacteria.

12. A culture of the strain is grown in the agar and dried.
A grid is drawn on the base of the Petri dish to mark out
different regions.
Inoculation of each square of the grid is done by a different
phage.
The phage drops are allowed to dry and are incubated:
The susceptible phage regions will show a circular clearing
where the bacteria have been.

13. A culture of bacteria infected by bacteriophages,
the "holes" are areas where the bacteria have
been killed by the virus.

14. Agglutination Reactions
Agglutination reactions may be used to detect either the
presence of antigen or antibody in a sample.
1. Direct agglutination reactions are used to diagnose
some diseases, determine if a patient has been exposed
to a particular pathogen.
2. Indirect agglutination is used in disease diagnosis.

16. Latex Agglutination Procedure
The latex agglutination test as a rapid diagnostic
slide test for Staphylococcus aureus.
Using protein-coated latex particles that are able to
detect the clumping factor (bound coagulase and
protein A) that causes the S. aureus to adhere to the
black latex particles, producing a visible
agglutination.

17. A negative reaction results in little or no agglutination and no loss of the
black background within 60 seconds.
A positive agglutination reaction usually occurs in 15 seconds by a clumping
together.
of agglutination.
Observe all slides for the presence or absence
Rotate the slide in a circular motion for 60 seconds.
Spread the mixture over the entire circle.
Using an applicator stick or sterile needle, spread one colony of each
organism in the reagent.
Place one drop of Staphylococcus latex reagent in the center of the circle on
the slide.

19. Salmonella Latex Agglutination
Widal test
The main principle of widal test is that if homologous
antibody is present in patients serum, it will react with
respective antigen in the reagent and gives visible
clumping on the test card and agglutination in the tube.
The antigens used in the test are “H” and “O” antigens
of Salmonella Typhi and “H” antigen of S. Paratyphi.
“O” antigen is a somatic antigen and “H” antigen is
flagellar antigen.

20. SEMI-QUANTITATIVE
METHOD
Pipette one drop of isotonic saline into the first reaction circle and then
place 5, 10, 20, 40, 80 ul of the test sample on the remaining circles.
Add to each reaction circle, a drop of the antigen which showed
agglutination with the test sample in the screening method.
Using separate mixing sticks, mix the contents of each circle uniformly
over the reaction circles.
Rock the slide gently back and forth, observe for agglutination
macroscopically within one minute.

22. Polymerase chain reaction (PCR)
• PCR amplifies a specific region of a DNA strand
(the DNA target).
• For detection of bacteria to strain level precisely.
• The principle of the method is simple; when a
pure PCR product of the 16SRNA gene is
obtained, sequenced, and aligned against bacterial
DNA data base, then the bacterium can be
identified.
• 16sRNA is highly conserved gene in the bacteria.

25. Thermal cycler

26. DNase Test
DNase Test is used to distinguish
Serratia species from Enterobacter
species, Neisseria species, and
Staphylococcus aureus from other
Staphylococcus species.
To aid in the
differentiation
between Klebsiella,
Enterobacter, and
Serratia spp.

27. The test organism is grown on an
agar medium containing DNA.
Following incubation, DNase
activity is determined by the
addition of 0.1% toluidine blue
to the surface of the agar.
DNase-positive cultures capable
of DNA hydrolysis will show a
rose-pink halo around the area
of growth.
The absence of this halo is
indicative of a negative result
and the inability of the organism
to produce Dnase.

28. The inoculum should be from an overnight
pure culture grown on Brain Heart Infusion
Agar, Sheep Blood Agar, or in BHI Broth.
Using a direct inoculum, streak the plate or
slants.
Incubate aerobically at 35°C.
Examine after 24 hours. If growth is poor
reincubate medium for an additional 24 hours
until good growth is observed.

29. A rose-pink halo around the area of growth on the left side of the
plate indicates a positive result, while the absence of a halo on
the right is a negative result.

30. Enzyme-Linked Immunosorbent Assay
(ELISA)
• A- indirect ELISA is used to
screen patients for the presence
of HIV antibodies, rubella
virus antibodies, and others.
• B- The direct ELISA may be
used to detect viral and
bacterial antigens.
ELISA Test
may be used
to detect the
presence and
amount of
either antigen
or antibody
in a sample.

31. Principle for ELISA
procedure is a widely accepted method that is used for the
detection of specific antigens or antibodies.
The procedure is predicated on the use of an enzyme-linked
(labeled) specific antibody to demonstrate the agglutination
reaction for the interpretation of the test result.
This test can be performed as a double-antibody technique or
as an indirect immunosorbent assay.

32. An enzyme-linked antibody, specific for the antigen, is now
added.
If the antigen is present in the well, this labeled
antibody binds to the antigen, forming an antibody-antigen-
antibody complex. Any unbound enzyme-linked antibody is
again removed by washing.
This is followed by the addition of a substrate that is capable
of producing a colored end product upon its reaction with the
enzyme.
The resulted enzymatically produced color change may be
observed by eye or spectrophotometrically.

33. The quantitative "reading" is usually
based on detection of intensity of
transmitted light by spectrophotometry,
which involves quantitation of
transmission of some specific
wavelength of light through the liquid
(as well as the transparent bottom of the
well in the multiple-well plate format).