This document discusses various methods used to identify unknown bacterial cultures, which is a major responsibility of microbiologists. It outlines staining techniques like Gram staining, acid-fast staining, endospore staining, and capsule staining. These techniques examine morphological characteristics of bacteria like shape, arrangement, presence of spores or capsules. The document also mentions biochemical tests that detect bacterial enzymatic activity or ability to ferment carbohydrates and produce acids/gases. Identifying pathogenic bacteria is important for medical diagnostics and food/brewing industries to prevent contamination.

1. BY:
MRS. MALI DHANASHRI R.
ASSISTANT PROFESSOR,
GES’S SIR DR. M. S. GOSAVI COLLEGE OF PHARMACEUTICAL
EDUCATION AND RESEACH

2.  Identification is the process of determining to which established taxon a new
isolate or unknown strain belongs.
 Identification of unknown bacterial culture is one of the major responsibilities of a
microbiologist
 Samples of blood, tissue, water, food and cosmetics are examined daily in
laboratories throughout the world for the presence of contaminants and
pathogenic microorganisms.
 Pharma industries and research institutes are constantly screening soil, water,
marine samaples to isolate new antibiotics, enzymes and vitamins producing
microorganisms.
 Bergey’s Manual of Systemic Bacteriology (BMSB) has been the official,
internationally accepted reference for bacterial classification.
 In the current edition of Bergey’s manual, bacteria is classified into 33 groups called
sections and contain information of 4000 bacterial species.

3. Importance of identification
 Medical diagnostics & clinical significance — identifying
a pathogen isolated from a patient.
 Food & brewage industries — identifying a microbial
contaminant responsible for food spoilage and
fermentation.
 Research setting — identifying a new isolate which
carries out an important process.

4. IDENTIFICATION METHODS
 The methods fall into three categories:
 Phenotypic/Morphology (micro and
macroscopic)
 Immunological/Serological analysis
 Genetic techniques/Molecular method

5. Staining Techniques
 To study size, shape, arrangement and properties and
different specific groups of microorganisms, Biological
stains are used.
 Stains is an organic compounds containing a benzine
rings with chromophore and auxochrome group.
 Different types of staining techniques are used to study
the morphological and structural properties of
microorganisms.

6. Types of staining

8. Dyes
Basic dyes:
 Methylene blue, Basic fuchsin, Crystal violet, Safranine, Malachite green.
 Have positively charged groups (usually from penta-valent nitrogen).
 Generally sold as chloride salts.
 Basic dyes bind to negatively charged molecules such as nucleic acids, many
proteins and surfaces of bacterial and archeal cells.
 Used for Positive staining

9.  Acidic dyes:
 Eosin, Rose Bengal, Nigrosin(indian ink), Congo red and Acid fuchsin possess
groups such as carboxyls (-COOH) and phenolic hydroxyls (-OH).
 Acidic dyes, in their ionized form, have a negative charge and bind to positively
charged cell structures.
 Used for negative staining
 Neutarl dyes:
 Both positively and negatively charged ions impart diiferent colour to different
component
 E.g. Leishman’s stain, Wrights stain.

10. Smear Preparation
 Microbial smear : It is a very small amount of microbial
growth spread on a clean slide and drying by air .
 The purpose of making a smear is to fix the bacteria onto the
slide and to prevent the sample from being lost during a
staining procedure.
 A smear can be prepared from a solid or broth medium.
 The preparation of a smear is required for many laboratory
procedures, including the Gram-stain.

11. Procedure
 Place one needle of solid bacterial growth or two loops of
liquid bacterial growth in the center of a clean slide.
 If working from a solid medium, add one drop (and only one
drop) of water to your specimen with a water bottle. If using a
broth medium, do not add the water.
 Now, with your inoculating loop, mix the specimen with the
water completely and spread the mixture out to cover about
half of the total slide area.
 Place the slide on a slide warmer and wait for it to dry. The
smear is now ready for the staining procedure.

12. Smear selection:
 If selected smear is embedded in paraffin,
 it is necessary to remove the paraffin so that a watery stain
may penetrate the bacterial cell.
 The paraffin is removed with xylene, the xylene is then
removed with alcohol and the alcohol is replaced with water.
 The staining is then done.

13. Smear fixation:
 Fixation of bacterial cell on slide.
 Heat Fixation:
 The process of passing the smear after drying several times over benzene burner
to fix the microbes on slide and prepare it for staining .
 Allow the slide to cool before staining
 Methanol Fixation:
 Flood the smear with methanol for one minute.
 Drain the slide and allow to dry.

14. SIMPLE STAINING/Monochrome
Stains
 Used only single stain
 Ex: Any one at a time- Methylene blue(2-3mins), Crystal violet(1-2mins),
Carbol fuchsin(15-30secs), Safranin(1mins), Malachit green(1-2mins),
etc.
 Used to study Size, shape and bacterial cell arrangements.
 Basic stains with a positively charged chromogens (any substance
that can become a pigment or coloring matter, as a substance in organic
fluids that forms coloured compounds when oxidized, or a compound, not
itself a dye, that can become a dye) are used.
 Purpose: To elucidate the morphology and arrangement of bacterial
cells.

16. PROCEDURE
 Select oil/grease free slide. Do it by washing with detergent and wiping
the excess water and then dry the slide by passed through flame.
 These slide is allowed to air dry and smear of sample is applied.
 After air drying these slide is rapidly passed through a flame for three to
four times for heat fixation.
 After heat fixation the slide is flooded with a particular stain and these
stain is allowed to react for two-three minutes.
 Further the slide is washed under running distilled water.
 The slide is air dried and watched under oil immersion microscope.

17. Mechanism behind the simple staining
 A stain has a ability to bind a cellular component .These abilities depend
upon the charges present on cellular component and charges present
on chromophore group of stain.
 Bacteria has large number of carboxyl group on its surface and these
carboxyl group has negative charge.
 When these carboxyl group carry out ionization reaction it shows COO– and
H+
Ionisation
COOH COO– + H+
 In nature, these H+ ions (unstable) are present on cell surface and further
replaced by other positively charged ions like Na+ or k+ and H+ bonds with
oxygen to form water.

19. NEGATIVE STAINING
 Colouring the Background of Object
 Not stain but made visible against dark background
 Bacteria are mixed with acidic stains such as Eosin or Nigrosin that provide a
uniformly coloured background against which the unstained bacteria stand out in
contrast.
 Useful to observed bacteria that are difficult to stain (Spirili & spirochetes–
Trepanoma palladium, Borrelia burgdorferi, Leptospira) and in demonstration
of bacterial capsule.
 Acidic stains has negative charge; therefore, it doesn’t combine with
negatively charged of bacteria cell surface.

20. Positive
staining
Negative
staining
Simple positive staining: all bacteria are
colored,
Simple negative staining: background is
dark, bacteria are without any color

21. Advantages over Simple:
1. Natural size and shape of microorganism can be seen
2. Heat fixation is not required
3. Doesn't need physical and chemical treatment.
4. It is possible to observe bacteria that are difficult to
stain.

22. Differential staining techniques
 The differential staining techniques are based on the
application of a set of several different dyes which react
differently with different types of microorganism.
 Hence, they can be used to distinguish among them.
 An example is the Gram staining technique.
 This procedure separates bacteria into two groups: Gram
positive bacteria and Gram negative bacteria.

23. GRAM STANING
 It is not only reveals the size and shape of bacteria but also used
to differentiate bacteria into Gram positive and Gram negative
cells.
 Hence, called differential staining.
 It is first and usually the only method employed for the
diagnostic identification of bacteria in clinical specimens.
 Provides more information about the characteristics of the cell wall
(Thickness).
 A stain is a chemical substance that adheres to a cell, giving the
cell colour.

24. Principle
 Dr. HANS CHRISTIAN GRAM:
 Danish bacteriologist noted for his development of the Gram stain in 1884.
 Violet dye and the iodine combine to form an insoluble, dark purple compound in the
bacterial protoplasm and cell wall.
 This compound is dissociable in the decolourizer, which dissolves and removes its two
components from the cell.
 But the removal is much slower from Gram-positive than from the Gram-negative bacteria,
so that by correct timing the former stay dark purple whilst the latter become colourless.
 The difference between the two types of bacteria is that the Gram positive have thicker and
denser peptidoglycan layers in their cell walls, which makes them less permeable to the
stain than those of the Gram negative bacteria.
 The iodine has a critical role in enhancing this difference.
 It seems to bind temporarily to the peptidoglycan and make it even less permeable to the
dye.

25. Procedure:
Thin smear of bacterial culture was made on clean
glass slide
Air dried and heat fixed
Smear was covered with crystal violet for 30 seconds
Slide was washed with distilled water
Smear was covered with Grams iodine solution for 60
seconds
Slide was washed with 95% ethyl alcohol and then
distilled water
Again the smear was covered with safranin for 30
seconds
Washed with distilled water and blot dried
Air dried and observed under microscope

26.  Gram-positive bacteria appear
blue or purple
 E.g. Staphylococcus aureus,
Streptococcus pyogenes,
Corynebacterium diphteriae
 Gram-negative cells will appear
pink to red
 E.g.Neisseria meningitidis,
Escherichia coli, Pseudomonas
aeruginosa

27. ACID FAST STAINING
 Another widely used differential staining procedure in
bacteriology developed by Ehrlich in 1882.
 Also known as Ziehl–Neelsen staining.
 Some bacteria(specially M. Tuberculosis, Nocardia
asteroides, Actinomycetes, and M. leprae) resist toward the
gram staining or other staining due to high lipid content in the
cell wall, hence they are called ACID FAST BACTERIA.
 5% aquouse phenol and heat(physical intensifier) are used to
enhance the penetration of primary stain(chemical intensifier)

28.  Some bacteria produce the waxy
substance mycolic acid when they construct their cell walls.
 Mycolic acid acts as a barrier, protecting the cells from
dehydrating, as well as from phagocytosis by immune system
cells in a host.
 This waxy barrier also prevents stains from penetrating the
cell, which is why the Gram stain does not work with
mycobacteria such as Mycobacterium, which are pathogens
of humans and animals.

29. Procedure:
 Place a drop of NaCl onto the glass slide.
 Using a sterilized and cooled inoculation loop, obtain a very small sample of a bacterial colony.
 Gently mix the bacteria into the NaCl drop.
 Let the bacterial sample air dry
 Using slide holder, pass the dried slide through the flame of Bunsen burner 3 or 4 times,smear
side facing up.
 Flood slides with Kinyoun carbolfuchsin (or Ziehl-Neelsen Carbol Fuchsin (ZNCF) for 5 minutes.
 Rinse gently with water until the water flows off clear.
 Flood slides with acid-alcohol (3% HCl in ethanol) for 3~5 seconds.
 Rinse gently with water until the water flows off clear.
 Flood slides with methylene blue for 1 minutes.
 Rinse gently with water until the water flows off clear.
 Allow slides to air dry before viewing

30.  acid fast bacteria appear
bright red
 E.g. Mycobacterium
tuberculosis,
Mycobacterium avium
 non-acid fast bacteria
appear blue
 E.g. Escherichia coli,
Staphylococcus aureus, etc.

31. Endospore Stain
 Endospores are dormant forms of living bacteria
 Mature endospores are highly resistant to environmental conditions such as heat and
chemicals.
 Because the endospore coat is highly resistant to staining, a special method was
developed to make them easier to see with a brightfield microscope.
 This method, called the endospore stain, uses either heat or long exposure time to
entice the endospores to take up the primary stain, usually a water soluble dye such
as malachite green since endospores are permeable to water.
 Following a decolorization step which removes the dye from the vegetative cells in
the smear, the counterstain safranin is applied to provide color and contrast.

32. o
 SCHAEFFER AND FULTON METHOD:
 Spore staining, Malachit green (primary stain) and
counter stain(safranin).
 Thin smear of bacterial culture was made on clean glass
slide
 Air dried and heat fixed
 Smear was covered with malachite green
 Slide was steamed for 5 minutes by adding more stain t
the smear for time to time
 Slide was washed slowly under tap water
 Again the smear was covered with safranin for 30
seconds
 Smear was washed with distilled water and air dried
 Observed under microscope

33.  Endospores can also be directly observed in cells by
using phase contrast microscopy

34. Capsule staining
 Bacterial capsules play a role in adherence, protection (they inhibit ingestion and
killing by phagocytes), securing nutrients, and cell-to-cell recognition.
 The capsule is non-ionic, so that the dyes commonly used will not bind to it. Two
dyes, one acidic and one basic, are used to stain the background and the cell
wall, respectively.
 Negative staining methods contrast a translucent, darker colored, background with
stained cells but an unstained capsule. The background is formed with india ink or
nigrosin or congo red.
 A positive capsule stain requires a mordant that precipitates the capsule. By
counterstaining with dyes like crystal violet, methylene blue or carbolfuchsin,
bacterial cell wall takes up the dye. Capsules appear colourless with stained cells
against dark background.

35. Procedure
 Using sterile technique, add a loopful of bacterial culture to tube with 1 ml
NaCl.
 Add one drop of carbolfuchsin into the tube and mix gently.
 Heat the mixture under flame 1min.
 Place a drop of mixture onto the glass slide.
 Place a drop of nigrosin onto the same glass slide next to drop of
mixture of bacteria and the dye.
 Use the other slide to drag the nigrosin-cell mixture into a thin film along
the first slide.
 Allow to air dry for 5-7 minutes (do not heat fix).
 Examine the smear microscopically (100X) for the presence of
encapsulated cells as indicated by clear zones surrounding the cells.

36.  Encapsulated bacteria: Clear halos (capsules) are
observed around pink bacteria against dark
background
 E.g. Bacillus anthracis, Klebsiella pneumoniae,
Streptococcus pneumoniae, Neisseria meningitidis,
Clostridium perfringens;
 Bacteria without capsules: pink bacteria with no
clear halos
 E.g.Neisseria gonorrhoreae, etc.

38. Biochemical Tests
 The microbe is cultured in a media with a special substrate and tested for an end
product.
 Prominent biochemical tests include:
Enzymes
Carbohydrate fermentation
Acid production
Gas production
Sensitivity to drugs

39. Biochemicals…. Enzymes
Catalase test
Oxidase test
Urease test
Coagulase test

40. CATALASE TEST
 Catalase is present in most aerobic and facultative anaerobic bacteria (except
streptococcus spp).
 Hydrogen peroxide forms as one of the oxidative end product of aerobic
carbohydrate metabolism.
 If this is allowed to accumulate in the bacterial cells it becomes lethal to the
bacteria
 Catalase thus helps in converting H2O2 to H2O and O2
 The presence of the enzyme in a bacterial isolate is evident when a small
inoculum is introduced into hydrogen peroxide and the rapid effervescence of
O2 bubbles occurs.

41. Procedure
 Nutrient agar medium was prepared
 The medium was poured into culture tubes and flasks
 It was sterilized by autoclaving at 151b pressure for 15 minutes
 The nutrient agar slants were inoculated with test organisms
 An inoculated nutrient agar slant was kept as control
 The cultures were incubated at 35°C and 3-4 drops of hydrogen
peroxide was added on the growth of each slant culture
 The culture was observed for the appearance or absence of
gas bubbles.

42. Positive
Micrococcus
Staphylococcus
Bacillus
Listeria monocytogenes
Enterobacteriacae
Gonococcus & Meningococcus
Vibriocholerae
Pseudo/Aero/Plesiomonas

43. OXIDASE TEST
 This test depends on the presence of cytochrome oxidase in
bacteria
Procedure- Place a piece of filter paper in petri dish and add 3
drops of freshly prepared oxidase reagent (1% solution of
tetramethyl-p-phenylene diamine)
Using a sterile glass rod or toothpick, remove a colony of test
organisms from a culture plate and smear it on the filter paper
Oxidase positive organisms give blue/ dark purple color within
5-10 seconds, and in oxidase negative organisms, color does
not change.

44.  Positive
Pseudomonas spp.
Aeromonas spp.
Vibrio spp.
Alcaligenes spp.
Neisseria spp.
Haemophilus sps

45. Coagulase test
 This test is used to differentiate Staphylococcus aureus (positive) from
coagulase negative Staphylococci
 When a bacterial suspension is mixed with plasma, this enzyme causes
alteration in fibrinogen.
 leading to precipitation on the staphylococcal cells, causing the cells to
clump.
 Slide test: Positive when there is Macroscopic clumping in 10 seconds
or less in a plasma drop and no clumping in a saline or water drop.
 Tube test: Positive when there is a Clot of any size

47. Urease test
Some bacteria produce urease an enzyme that hydrolyzes urea into
ammonia and carbon dioxide.
The test for urease production relies on the fact that the ammonia
produced upon hydroysis is alkaline.
The test organism is inoculated into a urea broth that contains phenol
red, a pH indicator, and has a pH of 6.8. At this pH phenol red is salmon
color. However, when the pH rises above 8.1 phenol red turns a cerise
(hot pink) color.
The urease test is useful for differentiating Salmonella which is urease
negative, from Proteus which is urease positive.

48.  Urease agar medium was prepared and
sterilized using autoclave
 The medium was allowed to solidify in the
slanting position to form a slope
 The slants were inoculated with test
organism
 The tubes were incubated at 37°C for 24
to 48hrs
 The slants were observed for colour

49. IMViC test:
 These are a group of individual tests used in microbiology lab
testing to identify an organism in the coliform group.
 Each of the letters in “IMViC” stands for one individual test.
 Indole: Break down the amino acid Tryptophan
 Methyl Red: Glucose oxidation
 Voges-Proskauer: Production of neutral end products
 Citrate: Citrate fermentation

50. The lower case "i" is merely for "in" as the Citrate test requires
coliform samples to be placed "in Citrate". Triple Sugar Test (TSI),
Urease Test and Malonate Test are also included in this test.
 Some microorganism are differentiate on the basis of enzyme-
catalysed metabolic reactions
 Presence and absence of certain enzymes, intermediately
metabolites end products often give valuable information in
identifying and classifying microorganism.
 These tests are useful in distinguishing members of
Enterobacteriaceae(Aerobic, non-acid fast, gram negative bacilli
found in human and other animal intestine.

51. Indol Production test
 Some bacteria posses tryptophanase enzyme.
 The enzyme is capable of converting tryptophan (an amino acid) to
indole and pyruvic acid, ammonia.
 Pyruvic acid can be converted to energy or used to synthesize other
compounds required by the cell.
 Indole reacts with added Kovac’s reagent to form rosindole dye which is red
in color (indole +).
 Principle Reaction:

52.  Indol production is detected by inoculating
the test microorganisms into peptone water
and incubating it at 37°C for 2-5days.
 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 indol.

53. Procedure:
 1% tryptone broth was prepared and sterilized using autoclave
at 151bs for 15 minutes
 The tryptone broth was inoculated with test organism and an
uninoculated tube was kept as control
 The tubes were incubated at 35°C for 48hours; 1ml of Kovac s
reagent was added 48hrs of incubation
 The tubes were shaken gently after intervals of 10 to 15minutes
 The tubes were allowed to stand for few minutes to permit the
reagent to come to the top.
 The tubes were observed for colour produced in the top layer

54. Methyl red (MR) Test
 Used to detect the production of acid during fermentation of glucose.
 Principle: This is to detect the ability of an organism to produce and
maintain stable acid end products from glucose fermentation.
 Some bacteria produce large amounts of acids from glucose fermentation that
they overcome the buffering action of the system.
 By production of acid, pH of the medium falls and maintained below 4.5
 Methyl red is a pH indicator (red at pH less than 4.4 and yellow at a pH greater
than 6).
 Methyl red test and Voges-Proskauer test both are done in methyl red–Voges-
Proskauer (MR-VP) broth, but the reagents that are added varies according to
the test.

55.  Mainly used to differentiate between E.coli and E. aerogen
 Principle Reactions:

56. Procedure:
 MRVP broth (Glucose phosphate peptone water)was
prepared and sterilized using autoclave
 5ml of the broth was poured into each tube
 The tubes were inoculated with test organism
 All the tubes were incubated at 25°C for 48hrs
 5 drops of methyl red indicator was added to the tubes of
each set
 The change in color of methyl red was observed for MR test
 A red colour indicates that glucose has been oxidized.
 Methyl red positive: Red colour (Acid production)- E.coli
 Methyl red negative: No colour (No acid production)- E.
aerogen

57. Voges-Proskauer Test
 Used to determine the ability of microbes to produce non-acidic or neutral end
products (acetyl methyl corbinol- acetoin, 2,3-butanediol and Diacetyl).
 Principle: Acetyl-methyl carbinol (acetoin) is an intermediate in the production of
butylene glycol. In this test two reagents, 40% KOH and alpha-naphthol are
added to test broth after incubation and exposed to atmospheric oxygen. If
acetoin is present, it is oxidized in the presence of air and KOH to diacetyl.
Diacetyl then reacts with guanidine components of peptone, in the presence of
alphanaphthol to produce red color. Role of alpha-naphthol is that of a catalyst
and a color intensifier.

58.  MR-VP broth is a combined medium used for two tests—Methyl Red
and Voges-Proskauer.
 This test is characterizes E.aerogen
 In the alkali medium and oxygen, the amount of acetyl methyl
carbinol present in the medium is oxidised to diacetyl which react
with the peptone of the broth to give a red colour.
 Principle Reactions:

59. Procedure:
 MRVP broth (Glucose phosphate peptone water)was
prepared and sterilized using autoclave
 5ml of the broth was poured into each tube
 The tubes were inoculated with test organism
 All the tubes were incubated at 25°C for 48hrs
 12 drops of VP reagent -1 and 2-3 drops of VP reagent-
II was added to the other set of tubes
 The tubes were gently shaken for 30 seconds with the
caps off to expose the media to oxygen
 The tubes were kept aside for 15-30minutes and
observed for change in colour for the VP test
 Klebsiella pneumoniae: Positive
 Escherichia coli: Negative

60. Citrate Utilization Test
 Used to determine if an organism is capable of fermenting citrate as the
sole source of carbon for growth.
 It indicated by the production of turbidity in the medium.
 The ability of an organism to utilize citrate occurs via the enzyme citrase.

61. Principle:
 Bacteria are inoculated on a medium containing sodium citrate and
a pH indicator bromothymol blue.
 Utilization of citrate involves the enzyme citritase, which breaks
down citrate to oxaloacetate and acetate. Oxaloacetate is further
broken down to pyruvate and CO2.
 Production of Na2CO3 as well as NH3 from utilization of sodium
citrate and ammonium salt respectively results in alkaline pH. This
results in change of medium’s color from green to blue.

62. Procedure:
 Simmon’s citrate agar media was prepared and
sterilized using autoclave
 5ml of media was poured into the culture tubes and
slants were prepared
 Simmon’s citrate agar slants were inoculated with
test organism
 The uninoculated tubes were kept as control
 The tubes were incubated at 370C for 48hours
 Slant culture was observed for the growth and
coloration of the medium

64. IMViC results
Species Indole Methyl Red
Voges-
Proskauer
Citrate
Escherichia coli Positive Positive Negative Negative
Shigella spp. Negative Positive Negative Negative
Salmonella spp. Negative Positive Negative Positive
Klebsiella spp. Negative Negative Positive Positive
Proteus vulgaris Positive Positive Negative Negative
Proteus mirabils Negative Positive Negative Positive
Citrobacter
freundii
Negative Positive Negative Positive
Enterobacter
aerogenes
Negative Negative Positive Positive

65. Triple sugar iron agar(TSI)
 This test is used to determine the ability to:
1- Reduce sulfur into H2S.
2 Lactose & /or Sucrose fermentation.
3 Glucose fermentation and Gas production.
 Used to differentiate among the different groups of
Enterobacteriaceae

66.  Media Used: Triple sugar-iron agar
 TSI contains 10 parts lactose: 10 parts sucrose: 1 part glucose and peptone.
 Sodium thiosulfate & ferrous sulfate (iron).
 Phenol red and ferrous sulphate serves as indicators of acidification and H2S formation,
respectively.
 The formation of CO2 and H2 is indicated by the presence of bubbles or cracks in the
agar or by separation of the agar from the sides or bottom of thetube.
 The production of H2S requires an acidic environment and is indicated by blackening of
the buttom of the medium in the tube.

67. Procedure and result interpretation
 Inoculate TSI medium with an organism by inoculating needle
by stabbing the butt & streaking the slant, incubate at 37°C for
24 hours.
 Alkaline slant/no change in the butt (K/NC) = Glucose, lactose
and sucrose non-utilizer (alkaline slant/alkaline butt)
 Alkaline slant/acid butt (K/A) = Glucose fermentation only.
 Acid slant/acid butt (A/A), with gas production = Glucose,
sucrose, and/or lactose fermenter.
 Alkaline slant/acid butt (K/A), H2S production = Glucose
fermentation only.

69. SIM- SULFIDE,INDOLE,
MOTILITY
 It is performed on sulfide-indole-motility (SIM) medium or
in tryptophan broth, or in motility urease indole (MIU)
medium.
 This is a differential medium. It tests the ability of an
organism to do several things: reduce sulfur, produce
indole and swim through the agar (be motile).
 SIM is commonly used to differentiate members of
Enterobacteriaceae.
 If sulfide is produced, a black color forms in the medium.
 Proteus mirabilis is positive for H2S production.

70.  Bacteria that have the enzyme tryptophanase,
can convert the amino acid, tryptophane to
indole. Indole reacts with added Kovac’s
reagent to form rosindole dye which is red in
color.
 Escherichia coli is indole positive.
 SIM tubes are inoculated with a single stab to
the bottom of the tube. If an organism is motile
then the growth will radiate from the stab mark
and and make the entire tube appear turbid.
 Pseudomonas aeruginosa and Proteus
mirabilis are motile.