1. Identification of Unknown Bacteria （Enteric Gram Negative Rods）
LAB EXERCISE 5
Identification of Unknown Bacteria
（Enteric Gram Negative Rods）
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3. Enterobacteriaceae Infections

4. Lactose fermenters (LF) E. coli Klebsiella Enterobacter ……
Enterobacteriaceae
Lactose fermenters (LF)
E. coli
Klebsiella
Enterobacter ……
Non-lactose fermenters (LNF)
Salmonella
Shigella
Proteus

5. Classification of Enterobacteriaceae
There are several selective and differential media used to isolate distinguishes between LF & LNF.
The most important media are:
MacConKey agar
Eosin Methylene Blue (EMB) agar
Salmonella Shigella (SS) agar
Triple Sugar Iron (TSI) agar
Kligler Iron Agar (KIA)

6. Differentiate between LF & LNF by Growth on SS agar
SS agar is selective & differential medium for Enterobacteriaceae.

7. Differentiate between LF & LNF by Growth on KIA slant

8. Unpathogenic
Pathogenic

9. SOME IDENTIFICATION TEST
Biochemical test
Carbohydrate Fermentation
Indole test (Enzyme test)
Other
Motility

10. KIA (Kligler Iron Agar) Test
The indicator is RED at alkaline pH, and YELLOW at acidic pH.
Interpretation:
Red slant /yellow butt → only glucose is fermented.
Yellow slant /yellow butt →glucose and lactose both are fermented.
Bubbles or crack present → gas production.
Black precipitate present → H2S production.

11. LNF
H2S produced LF
Gases produced
Control
LNF

12. Carbohydrate Fermentation
Aim: To determine the ability of microbe to ferment a specific carbohydrate with or without the production of gas.
Material:
Carbohydrate
Glucose (Red cap) /golden
Mannitol (White cap)
Lactose (Yellow cap)
Sucrose (Black cap) /green
Maltose (Blue cap)
* Indicator: Phenol Red (acid → yellow)

13. Carbohydrate Fermentation
Interpretation:
Acid production: Changes the medium into yellow color- organism ferments the given carbohydrate and produce organic acids there by reducing the pH of the medium into acidic.
Acid and Gas production: Changes the medium into yellow color. Gas production can be detected by the presence of small bubbles in the inverted Durham tubes.
Absence of fermentation: No change observed in the colour of medium. The organism cannot utilize the carbohydrate but the organism continues to grow in the medium using other energy sources in the medium.

15. Indole Test
Aim: To determine the ability of microbe to degrade the amino acid tryptophan.
Interpretation:
Development of cherry red colour at the interface of the reagent and the broth, within seconds after adding the Kovacs’ reagent indicates the presence of indole and the test is positive.
If no colour change is observed, then the test is negative and so organisms are not capable of producing tryptophanase.

16. Motility Aim: To determine the ability of microbe to migrate (motile).
Interpretation:
Organism growing only in line of inoculation → non-motile.
Organism appears as haze beyond line of inoculation → motile

17. Serological test Object of study : Materials and equipment：
Use known antiserum (antibodies) to identify unknown antigens.
Materials and equipment：
1:20 Typhoid serum
1:20 Shiga serum

18. Serological test Practise：
Using a wax marker, draw 3 circles on 2 clean glass slides. Label the circles A, B, and C.
Add 1 drop of NS to the “A” circle, 1 drop of known Typhoid serum to the “B” circle, and 1 drop of known Shiga serum to the “C” circle.
Emulsify one or two colonies on each drop to make a smooth suspension.

19. Serological test Interpretation:
Agglutination of the bacteria indicates a positive reaction.
No agglutination is negative.

20. Widal test Object of study : Materials and equipment：
This is a tube agglutination test employed in the serological diagnosis of enteric fever.
Materials and equipment：
S.TYPHI "O" antigen
S.TYPHI "H" antigen
S.PARATYPHI "AH" antigen
S.PARATYPHI "BH" antigen
Patient serum (1:10 dilution)
200μL pipettor, Pipettor tips,
40-well Immunoplate

21. Widal test
Practise：
An immunoplate having 40 wells in 4 rows is taken. The rows are labeled as O, H, AH, BH.
Using a micropipettor add 50μL normal saline to all the wells in the 40-well immunoplate.
Add 50μL diluted patient serum (1:10) into well O1 containing 50μL saline, mixed by sucking and pumping the fluid with tip for 3 times.

22. Widal test
Practise：
Transfer 50μL from well O1 to O2 , mixed by gently pipetting. Use the same tip to transfer 50μL from well O2 to O3. Avoid bubbles. Using the same tip, repeat these twofold dilution down the entire row, discarding 50μL from A9 so that it ends up with 50μL of diluted serum. The dilution from well A1 to A9 is 1:20, 1:40……1:1280.

24. Widal test
Practise：
Add 50μL antigen S.TYPHI "O" to each well (from “9” to “1”), mix well. The final dilution from well O1 to O9 is 1:40, 1:80……1:2560; Well “10” is the control tube.
Repeat this doubling dilution with different antigen (H /AH /BH) in defined rows.
Mix each well and incubate for 1h at 45℃. And place the plate at RT standing for 15 minutes to wake up.

25. Widal test
Practise：
Observe the results and determine the titer of the serum ( + + ) .The highest dilution of serum causing obvious agglutination of bacteria ( + + ) is defined as the titer of the serum.

26. Widal test
Results：
First observe the control well 10. They should show a uniform turbidity in contrast to the aggregates seen in the wells containing the serum dilutions . it is represented as “ - ”.
The agglutination is represented as“+”:
“ ”represents all of the bacteria agglutinated. Big agglutin appears at the bottom of the tube, while the supernatant is clear.
“+ + +”represents part of the bacteria agglutinated. Middle agglutin appears at the bottom of the tube, while the supernatant is a little turbid.
“+ +” represents small part of the bacteria agglutinated. Half of the supernatant is turbid.

27. Widal test Results： Attentions The agglutination is represented as“+”:
“+”represents very small amount of the bacteria agglutinated, while the supernatant is turbid.
“ - ”represents no agglutination. The phenomenon is same as that of the control tube.
Attentions
Gently manipulate the pipettes. Do not break the test tubes with the pipettes. Be careful when you add and transfer the samples one by one, to avoid confusion.
Do not shake the test tubes when you observe the results , to avoid the spreading of the agglutinates during observation.

28. Interpretation of results
O<1:80,TH<1:160, PH<1:80
Normal value
O ≥1:80 & TH ≥1:160 or
O ≥1:80 & PH ≥1:80
Typhoid fever
Paratyphoid fever
O ≥1:80 & TH <1:160 or
O ≥1:80 & PH <1:80
Early infection or other salmonella infections
O <1:80 & TH ≥1:160 or
O <1:80 & PH ≥ 1:80
Vaccination or nonspecific memory reaction