1. Some Biochemical Tests Used In Bacterial Diagnosis (Identification)
Faculty of Medicine and Health Sciences
Microbiology Lab 12
Some Biochemical Tests Used In Bacterial Diagnosis (Identification)
Dear Students: although this file has 62 slides, 25 of them are only illustrating images and figures. Most of the information in the other slides are just explanation that requires you to understand the main idea and to memorize every thing
First Semester
Prepared by : Dr Alaeddin Abuzant, PhD Microbiology and Immunology

2. I-Biochemical Test Based on Carbohydrate Utilization:
In general, when it comes to carbohydrates (CHO) , a bacterium either
1- Ferments CHO ( anaerobic and facultative anaerobic bacteria)
2- Oxidizes CHO ( some aerobic bacteria)
3- Both (Fermentation and oxidation)( facultative anaerobic bacteria)
4- None ( in this case, the bacterium does not utilize CHO at all)

3. Tests Based on CHO Fermentation:
CHO fermentation by bacteria results in:
1- The production of acidic products such as lactic, acetic acid and pyruvic acid) 2- In addition to acidic products, some bacteria are able to produce gas such as H2 and CO2)
Carbohydrate utilization tests based on CHO fermentation, are designed to detect the production of acidic products produce as a result of the fermentation process. Detection of these acidic compund can be easily done by using pH indicators.
Ph indicators are chemicals that change their colors according to the change in pH (colors in acidic and alkaline pH)
Example: Phenol red has a red color in alkaline to neutral pH and a yellow color in acidic pH .

4. Phenol Red Broth Test:
It is a simple test that is used to detect the fermentation of a particulate sugars as well as the production of gas by the tested bacterium
In this test, a tube that contains Phenol Red Broth is utilized in the experiment.
Phenol Red Broth contains a particular SINGLE sugar ( to be tested whether this sugar can be fermented by the test bacterium) and a pH indicator ( Phenol Red)
Phenol Red Broth is inoculated aseptically by the bacterium to be tested and incubated for an over night at 37 C.
Upon the fermentation of the sugar included in the Phenol Red Broth by the test bacterium, the generated acidic product lower the pH of the broth so that the color of the broth changes from red to yellow ( this will be seen after an over night incubation).
If the tested bacterium does not, you will not notice a change in the color of the broth

5. If the fermentation of the included sugar by the tested bacterium also generates gas, gas production is detected by including a small tube known as (Durham tube) that is placed in the test tube (larger tube) of the test in an inverted manner.
Upon gas production due to the fermentation process of the included sugar during the incubation period by the test bacterium, the generated gas accumulates in the Durham tube to replace the broth ( completely or partially) found in this tude (Durham tube).
Accordingly, by the end of the incubation period of the test, Durham tube appears empty.
Phenol Red: is a pH indicator which turns yellow below a pH of 6.8 and fuchsia to Red above a pH of 7.4.

6. Neutral/Alkaline pH Acidic pH
Phenol Red Broth
Neutral/Alkaline pH Acidic pH

8. The tested bacterium ferments the included sugar WITHOUT gas production

9. The tested bacterium ferments the included sugar WITH gas production

11. THREE bacterial species X, Y and Z were inoculated individually (under a septic conditions) into Phenol Red Broth tubes that contains ( Say Glucose) (all the tubes contain glucose)
After 24 h of Incubation
Results:
Bacterium X: ferments glucose with gas production
Bacterium Y: ferments glucose WITHOUT gas production
Bacterium Z: does not ferment glucose

12. A SINGLE bacterial species was inoculated into three Phenol Red Both tubes, EACH of which contains a PARTICULAR sugar
After 24 h of Incubation
Results:
Bacterium X: ferments glucose with gas production
Bacterium X: ferments sucrose WITHOUT gas production
Bacterium X: does NOT ferment lactose

13. What do you conclude about E coli ?
After 24 h of Incubation

14. What do you conclude about Proteus ?
After 24 h of Incubation

15. MRVP (the Methyl red-Vogues Proskauer test):
Certain types of bacteria are able to convert the acid produced during the fermentation into a neutral compound ( such as acetyl methyl carbinol ). In such cases, the usage of the pH indicator will NOT BE USEFUL
Accordingly, if the result of the previous test was negative (Phenol Red Broth),
do not jump into conclusion that the bacterium does not ferment the tested sugar. Maybe, it does so, but in converts the acid into a neutral compound that cannot be detected by the pH indicator……
But IT CAN BE DETECTED BY the Vogues Proskauer test.

16. Methyl red-Vogues Proskauer test : This test has two parts:
1- The MR portion (methyl red) is used to determine if the tested bacterium ferments glucose and the by a process that produces acidic compounds or NOT
2- The VP portion is used to determine whether tested bacterium ferments glucose to generate neutral compounds or NOT glucose.( in this case, the bacterium converts the acidic compounds produced by fermentation into neutral compounds…such as such as acetyl methyl carbinol (in this case pH indicator will not work)
To detect the presence acetyl methyl carbinol, a special test is used, which is the Vogues Proskauer test

17. Procedure of MRVP Test:
The MRVP tests are performed by inoculating a single tube of MRVP media with the bacterium to be tested
Then, the inoculated tube is for 3-5 days.
After the culture is grown, about half of the culture is transferred to a clean tube.
One tube of culture will be used to perform the MR test, the second tube is used to perform the VP test.

19. The MR (methyl red) test:
Aims to examine whether the tested bacterium ferments glucose with the production of acids
Few drops of Methyl red pH indicator is added to the MR tube:
A red color indicates a positive result (glucose can be converted into acidic end products.
A yellow color indicates a negative result.( this pH indicator has a yellow color in neutral/alkaline pH and a Red Color in acidic pH o the colors ……..
Note: ( this is opposite to the colors of the phenol red pH indicator used in the previous test ….Red in neutral/alkaline pH…Yellow in acidic pH)

21. If when adding few drops of the methyl red indicator to MR test tube you obtained a yellow color…….( means no acidic compound(s) is/are present……
We MUST NOT jump to conclusion that the tested bacterium does ferment glucose…..we have to test the other possibility which is that the tested bacterium may ferment glucose but it generates neutral compounds instead of acidic compounds….(pH indicator is not useful in this case)
We can examine for this possibility by conducting the other part of the test… the Vogues Proskauer test used for detecting the presence of neutral compounds such as acetmethylcarbinol (in this case pH indicator will not work)

22. VP (Vogues Proskauer) test:
This test is performed by adding few drops of VP reagents to the VP tube
VP reagent consists of:
Barritt’s reagent A that contains alpha-napthol
Barritt’s reagent B that cosists of potassium hydroxide (also)
Initially, few drops of reagent A is added followed by few drops of reagent B
Wait for about 15 minutes for color development to occur.

23. If acetyl methyl carbinol is presents: the color of broth color will turns into a reddish color (positive result)...This means that the tested bacterium ferments glucose but it generates neutral compound instead of acidic products ….
If acetyl methyl carbinol is NOT present: the broth remains yellowish (a negative result).
Voges-Proskauer Test Results
Positive Negative

24. Expected Results of MRVP tests:
A tested bacterium can be either
1- MR positive but negative for VP ….ferment glucose with acid production
2- MR negative but VP positive…..ferment glucose with neutral compound production
3- MR negative and VP negative ( both tests are negative) ..this implies that the tested bacterium does not ferment glucose at all ( no acidic or neutral compounds are present)
4- MR positive and VP positive ( very rare to be found……) implies that the tested bacterium ferments glucose and produce both acidic and neutral compounds)

25. Triple Sugar Iron Agar:
Triple sugar iron agar (TSI) is a differential medium that contains lactose (10 X) , sucrose (10 X) , a small amount of glucose ( 1X)
It also contains ferrous sulfate, and the pH indicator (Phenol Red)
It is used to differentiate enteric bacterium based on the ability to reduce sulfur and ferment carbohydrates. 
Triple sugar Iron (TSI) test examines:
Fermentation of glucose alone, glucose and lactose/sucrose, or three of them
Hydrogen sulfide production
Gas production

26. Triple Sugar Iron Agar:

27. Basically the pH indicator will change the color of the red medium in response to fermentation due to acid production. The color will change from Red to Yellow in the presence of acidic compounds
The presence of a black color precipitate indicates that H2S was produced. H2S reacts with the ferrous sulfate in the media to make ferrous sulfide, which is a black product.
Cracking in the medium indicates gas production

28. Procedure:
To inoculate the TSI tube with the bacterium to be tested, a sterile needle is used to stab the TSI agar in the tube and then uses a loop to streak the top slated region.
The tube is incubated for 18-24h for 37 hour and then examined

30. Expected TSI Results

33. Simmons Citrate Utilization Test:
This test is part of the IMViC (Indole, Methyl Red, Vogues Proskauer and Citrate)
These tests and is helpful in differentiating (diagnosis) of members of the Enterobacteriaceae .
Simmons citrate agar contains:
Sodium citrate as the sole source of carbon
Ammonium dihydrogen phosphate as the sole source of nitrogen,
A pH indicator (Bromthymol blue).

34. A bacterium which utilizes citrate as its sole carbon source use the enzyme citrase (citrase is citrate-permease which is a transporter that transports the citrate from the surrounding environment into the bacterial cell).
 Once inside the bacterial cell, citrate (an intermediate of the Kreb’s cycle) is converted into oxaloacetate (another intermediate of the Kreb’s cycle) in order to generate energy and other molecules. This will allow the bacteria to grow.
In addition, the citrate agar contains ammonium dihydrogen phosphate. If the tested bacterium can utilize citrate ( not all species of bacteria can do so) as a sole source of carbon, the growing bacterium will convert the ammonium dihydrogen phosphate in the test medium to ammonia and ammonium hydroxide.
Citrate agar is green in color. The basic compounds (have alkaline pH) that result due to citrate utilization convert the color of the pH indicator ( bromthymol blue) into a blue color
Note: At a neutral pH, bromthymol blue is green. At pH 7.5 or above, bromthymol blue turns blue
Note: The bacterium that cannot utilize citrate a sole source of Carbone will not be able to grow on the medium so that there will be no change in the color of the citrate agar by the end of the incubation period

35. . 
Procedure:
The slant of Simmons Citrate agar in the tube is inoculated with the bacterium to be tested
Note: the bacterium to be tested is only inoculated on the slant of Simmons Citrate agar because citrate utilization occurs under aerobic conditions.
The inoculated tube in then incubated for 24h
After 24 h of incubation, if the inoculated medium turns blue, the bacterium is citrate positive.  If there is no color change, the color of the medium remains green, the bacterium is said to citrate negative

37. After about 24h of incubation at 37C
Green (Negative) Blue (Positive)
Examples
Escherichia coli: Negative
Klebsiella pneumoniae: Positive

38. Starch Hydrolysis Test:
This test is used for the detection of the enzyme alpha amylase producing bacteria. This enzyme is used by bacteria to degrade polysaccharides such as starch
The test utilizes starch agar which is a simple nutritive medium with starch added. 
The cultured bacterium is positive for this enzyme ( produce alpha amylase), starch in the agar is d by this enzyme.
Starch can be detected easily by iodine. Upon the addition of iodine to the starch..the starch color is turned into purple.

39. Procedure:
A starch agar plate is inoculated with the bacterium to be tested and then the plate is incubated at 37 C
After 24 h of incubation, iodine is added to the plate
If the grown bacterium produces alpha amylase, all the surface of the culture medium will become purple except the area around the colonies ( growth) of the grown bacterium..it will remains white..
This is because the starch around the grown bacterium has been hydrolyzed because the grown bacterium produces the alpha-amylase enzyme , so no starch is left in these area to react with iodine and give a purple color.

41. II-Biochemical Test Based on Protein and Amino acid utilization

42. Gelatinase Test
Nutrient gelatin is a differential medium that tests the ability of a bacterium to produce gelatinase enzyme that hydrolyzes gelatin.
Gelatin is a protein derived from connective tissue. Gelatinase allows the bacterium that produces it to break down gelatin into smaller polypeptides, peptides, and amino acids that can cross the cell membrane and be utilized by the bacterium
Gelatin at a temperature below 32°C, it is a semisolid material. However, at temperatures above 32°C, it is a viscous liquid.
When gelatin is broken down, it can no longer solidify.  If a bacterium can break down gelatin, the areas where the bacterium has grown will remain liquid even if the gelatin is refrigerated.

43. Procedure: Nutrient Gelatin Deep Stab Method
The gelatin deep stab method employs nutrient gelatin tubes that contain 12% gelatin.
A heavy inoculum from a pure culture of the bacterium to be tested is deeply stabbed into the medium using a sterile needle .

44. The gelatin media is incubated for at least 48 hours, and then placed into the refrigerator for approximately 30 minutes.
If the gelatin is still intact (the bacteria did not produce gelatinize), the media will solidify in the refrigerator and a negative test result is recorded.
If the organism has produced sufficient Gelatinase, the tube will remain liquid (at least partially) and not solidify in the refrigerator. A positive test result is recorded
You can simply detect this, by tilting the tubes once taken out the refrigerator

45. Positive Negative Positive: Serratia marcescens
Negative: Salmonella typhimurium

46. Urease Test:
This test is used for the detection of the production of the urease enzyme in bacteria
Urease medium is a differential medium that tests the ability of bacterium to produce the urease enzyme.
The test medium contains urea and a very small amount of nutrients to support bacterial growth. In addition, it contains the pH indicator phenol red.  The Phenol red pH indicator turns yellow in an acidic environment and fuchsia in an alkaline environment. 
The test medium, urease agar is inoculated with the test bacterium and then incubated at 37 C for 18-24h
In case the tested bacterium was a urease-producer, during the incubation time, the urease enzyme of the growing bacteriaum hydrolyzes urea to ammonia and carbon dioxide.  Ammonia has an alkaline pH that turns the color of the medium from yellow to fuchsia )

47. Proteus, Helicobacter pylori and Klebsiella pneumoniae are among the urease producing bacteria ( are said to be urease positive

49. SIM Medium
SIM medium is a combination differential medium that tests three different parameters, which are represented by the three letters in the name. S I M stand for
Sulfur Reduction……S
Indole Production…..I
Motility………M

50. The sulfur reduction test is useful in differentiating members of enteric bacteria
The indole test is a component of the IMViC test ( Indole, MR, VP and Citrate), which is used for differentiating ( diagnosis) of species of Enterobacteriaceae. 
The motility test is useful for testing a wide variety of bacteria for motility
Note: SIM is very useful for differentiating Salmonella and Shigella.

51. SIM medium contains:
it is a semisolid medium, that contains the following
1-Nutrients needed to support bacterial growth : (one of the nutrients is peptone, which contains amino acids, including tryptophan).
2- Iron
3- Sodium thiosulfate.

52. Procedure:
An SIM tube is inoculating with the bacterium to be tested using a sterile needle
The needle is stabbed into SIM medium straight down approximately two-thirds of the depth of the media.
The needle is then pulled out of the medium straight up (as mush as possible).
The inoculated SIM tube is then incubated for approximately 24 hours.

53. Motility Test :
Motile bacterium possess flagella and has the ability to swim. Such bacteria are said to be motile.
After 24h of incubation, if the tested bacterium is motile, The SIM medium will or have fuzzy, diffused growth at the edges of the strapping line.
If the tested bacterium is not motile, it will show a line of growth in the SIM along the stapping line. The rest of the SIM medium will be clear.

54. S. aureus E. coli

55. Reduction of Sulfur to hydrogen sulfide:
If the tested bacterium can reduce sulfur to hydrogen sulfide, the hydrogen sulfide will combine with the iron to form ferric sulfide, which is a black precipitate.( similar to TSI test)
If there is any blackening of the medium, it indicates the reduction of sulfur and is a positive result.
Note: The sulfur and motility test results should be determined before you perform the indole test.

57. The Indole Test:
Some bacteria produce the enzyme tryptophanase, which hydrolyzes the amino acid tryptophan into indole, pyruvic acid, and ammonia.
The detection of indole is used as an indication for the production of the tryptophanase by the tested bacterium
The Kovac’s reagent is used to detect the presence of indole in SIM medium after 24 h of incubation.
The reagent colorless. It contains hydrochloric acid, p-dimethylaminobenzaldehyde (DMABA), and n-amyl alcohol.

58. A bout 1 ml of this reagent is added to the SIM medium
A bout 1 ml of this reagent is added to the SIM medium . If indole is present, it will react (DMABA) to produce a red quinoidal compound. 
The presence of indole indicates the tested bacterium produces tryptophanase enzyme ( positive)
So, if the reagent turns red, the indole test is positive..the tested bacterium is positive for tryptophanase ( it produces tryptophanase)
If it remains colorless, the indole test is negative…the tested bacterium is negative for tryptophanase ( it does not produce tryptophanase )
Example:
E coli Indole positive
Klebsiella pneumoniae indole negative

59. Klebsiella pneumoniae.
Klebsiella pneumoniae
indole negative
E coli
Indole positive