This document provides information about identification of bacteria through staining and biochemical tests. It discusses various staining techniques like simple staining, Gram staining, negative staining and acid fast staining. It explains the principles, procedures and results of these staining methods. It also describes the IMViC tests, which are used to identify Gram negative bacteria based on their ability to produce indole, change the color of methyl red, produce acetoin in Voges-Proskauer test and utilize citrate as a carbon source. Understanding these staining techniques and biochemical tests is important for identification of microbes.

1. MODERN COLLEGE OF PHARMACY
MOSHI, PUNE
Savitribai Phule Pune University
B.PHARM
Semester- III
By
Mrs. Sneha K. Patil
Assistant Professor
Dept. of Pharmaceutics
Identification of bacteria
(staining and Imvic test)

2. UNIT OUTCOMES
• To understand principle and procedure of staining
techniques
• To know the importance of biochemical tests in
identification of microbes with reference to Imvic
test
1

3. CONTENT
1. Introduction
2. Classification of stain
3. Staining techniques classification
4. Smear preparation and fixation
5. Simple staining
6. Gram staining
7. Negative staining
8. Acid fast staining
9. Biochemical test
10.References
2

4. 1. Introduction
3
 Staining is technique used in microscopy to enhance contrast in the
microscopic image
 Bacteria have nearly the same refractive index as water therefore,
when they are observed under a microscope they are opaque or
nearly invisible to the naked eye
 Different types of staining methods are used to make the cells and
their internal structures more visible under the light microscope
 Stains are frequently used in biology and medicine to highlight
structures in biological tissues for viewing, often with the aid of
different microscopes

5. 2. Classification of stain
4
 Stain is an organic compound containing a benzene ring with
chromophore and auxochrome group
 Classification
1. Acidic stain - Negatively charge, e.g. eosin, nigrosin, indian ink,
malachite green, acid fuchsin
2. Basic stain - Positive charge, e.g. Haematoxillin, methylene blue,
crystal violet, gention violet, safranin
3. Neutral stain - Both positively and negatively charged imparts
different colors to different components, e.g.
Geimsa’s stain, leishman’s stain, wright’s
stain

6. 3. Staining techniques classification
5
 Positive staining
 Where the actual cells are themselves colored and appear in a
clear background
(a) Simple staining: A stain which provides color contrast but gives
same color to all bacteria and cells, e.g. Loeffler’s methylene blue,
Polychrome methylene blue, Diluted carbol fuchsin
(b) Differential Staining: A stain which imparts different colors to
different bacteria is called differential stain(which contains more
than one stain), e.g.Gram’s stain , acid fast staining, special stains.

7. Staining techniques classification continue…..
6
 Negative staining
 Where the cells remain clear (uncolored) and the background is
colored to create a contrast to aid in the better visualization of the
image, e.g. Indian ink, nigrosin
Fig. 1 Negative staining

8. 4. Smear preparation and fixation
7
 Smear preparation
 Smear - is a distribution of bacterial cells on a slide for the purpose
of viewing them under the microscope
 Method
1. Aseptically a small sample of the culture is spread over a slide
surface
2. This is then allowed to air dry
3. The next step is heat fixation to help the cells adhere to the slide
surface
4. The smear is now ready for staining

9. 8
 Smear fixation
a. Heat fixation
1. Pass air-dried smears through a flame two or three times and do
not overheat
2. Allow slide to cool before staining
b. Chemical fixation
1. Chemical fixatives (chemicals causing fixation) generate chemical
bonds between proteins and other substances within the sample,
increasing their rigidity e.g. Formaldehyde, ethanol, methanol,
picric acid
2. Place air-dried smears in a coplin jar with methanol for one minute
and alternatively, flood smear with methanol for 1 min.

10. 5. Simple/ monochrome staining
9
 Simple Staining is a technique that only uses one type of stain on a
slide at a time
 Purpose – To determine the morphology and arrangement of cells
 Bacterial nucleic acid and certain wall components carry a negative
charge that strongly binds to the cationic chromogen (+)
 Principle
1. The surface of bacterial cell has acidic characteristics due to acidic
amino acids but on ionisation of carboxyl group ,imparts negative
charge to the cell

11. Simple staining continue….
10
Principle
2. In nature H+ is replaced by another positively charged ion e.g. Na+ /
K+ and H+ bonds with water to oxygen to form water
3. Thus, surface of an unstained bacterial cell is represented as shown
below
4. Basic dyes are available as a salts of acid

12. Simple staining continue….
11
 Procedure
1. Make a thin smear on a slide
2. Heat fixes the smear by passing the slide 2-3 times gently over the
Bunsen flame with the smear side up
3. Pour methylene blue over the smear and allow it to stand for 3
minutes
4. Wash the stained smear with water and air dry it
5. Observe the smear first under low power (10x) objective, and then
under oil immersion (100x) objective
6. Observe the presence of organisms and also the cellular content of
sample

13. 12
Simple staining continue….
Fig. 2 Procedure of simple staining

14. 13
Fig. 3 Simple staining

15. 6. Gram staining
14
 Gram staining is most widely used differential staining in
Microbiology for differentiation and identification of bacteria
 Gram staining differentiates the bacteria into 2 groups:
1. Gram positive
2. Gram negative

16. Gram staining continue…..
 The Gram stain was devised by the
Danish physician, Hans Christian
Joachim Gram, while working in
Berlin in 1883
 He later published this procedure in
1884
 At the time, Dr. Gram was studying
lung tissue sections from patients
who had died of pneumonia
15
Hans Christian Joachim Gram

17. Principle
16
 The Gram Reaction is dependent on permeability of the bacterial
cell wall and cytoplasmic membrane, to the dye-iodine complex
 In Gram positive bacteria, the crystal violet dye –iodine complex
combines to form a larger molecule which precipitates within the
cell
 Also the alcohol/acetone mixture which act as decolorizing agent,
cause dehydration of the multi-layared peptidoglycan of the cell wall
 This causes decreasing of the space between the molecules causing
the cell wall to trap the crystal violet iodine complex within the cell.
Hence the Gram positive bacteria do not get decolorized
and retain primary dye appearing violet
Gram staining continue…..

18. Principle
17
Gram staining continue…..
 Also, Gram positive bacteria have more acidic protoplasm and hence
bind to the basic dye more firmly
 In the case of Gram negative bacteria, the alcohol, being a lipid
solvent, dissolves the outer lipopolysaccharide membrane of the cell
wall and also damage the cytoplasmic membrane to which the
peptidoglycan is attached
 As a result, the dye-iodine complex is not retained within the cell
and permeates out of it during the process of decolourisation.
Hence when a counter stain is added, they take up the colour of the
stain and appear pink

19. Method
18
Gram staining continue…..
 Method consists of four components:
1. Primary stain- Crystal violet, Methyl violet & Gentian violet
2. Mordant- Gram Iodine, Rarely Lugol’s Iodine
3. Decolourizer - Alcohol,Acetone, Alcohol: Acetone (1:1) mixture
4. Counter stain - Dilute Carbol fuchsin, Safranin, Neutral red, Sandi
ford stain for Gonococci

20. Procedure
• Cells stain purple
• Cells remain purple
• Gram-positive cells remain
purple. Gram negative cells
become colourless
• Gram positive cells remain
purple. Gram-negative cells
appear red
19
Gram staining continue…..
 Step 1- Crystal violet
(primary stain) for 1 minute
& water rinse
 Step 2- Iodine (mordant) for
1 minute. Water rinse
 Step 3 – Alcohol
(decolourizer) for 10-30
seconds & water rinse
 Step 4 - Safranin
(counterstain) for 30-60
seconds, Water rinse and
blot dry

21. 20
Fig. 4 Gram staining procedure

22. 7. Negative staining
21
Principle
 Bacterial suspension is mixed with acidic stain (-ve charge)
e.g. eosin, nigrosin, congo red, rose bengal stain
 Acidic stain does not penetrate the bacterial cells because of the
negative charge on the surface of bacteria
 It forms a deposit around the cell, resulting in appearance of
bacterial cell colourless against dark background

23. Negative staining continue….
22
Procedure
1. A loopful of acidic stain was placed on one end of clean slide
2. A loopful of inoculum was transferred into the drop of stain
3. By using a second dirt-free slide with smooth edge over the
suspension, it was spread uniformly along the edge
4. The suspension was spread to the end of the slide so as to form a
uniform smear
5. The slide was then air dried and observed under oil immersion
objective
6. Colourless bacteria are seen against a dark background

24. Negative staining continue….
23
Advantages over positive staining
1. Heat fixation is not required for the cells
2. Natural size and shape of microorganisms can be seen
3. It is possible to observe bacteria that are difficult to stain e.g.
Spirilli
Application
1. Demonstration of bacterial capsule

25. 8. Acid fast staining/ Ziehl – Neelsen staining
24
 It is differential staining procedure widely use in bacteriology
 Acid fast stain was developed by Paul Ehrlich in 1882
 Ziehl and Neelsen independently proposed acid fast stain in 1882-
83, which is commonly used today
 Acid fast microorganisms – Some bacteria resist decolourisation by
both acid and alcohol (3% HCl + 95% ethanol)
 This technique differentiate bacteria into acid fast and non-acid fast
 It is extensively used in the diagnosis of M. tuberculosis and M.
leprae

26. Acid fast staining continue….
25
Principle
 Acid fastness of certain mycobacteria /Nocardia species is
correlated with their high lipid content (60% w/w) i.e. mycolic acid
 Due to high lipid content of cell wall, acid fast cells have relatively
low permeability to dye and hence it is difficult to stain
 Penetration of dye is facilitated with use of 5% aq. Phenol which act
as chemical intensifier
 Heat is also used as a physical intensifier
 Once these cells are stained, it is difficult to decolourise with acid
and alcohol

27. Acid fast staining continue….
26
Three reagent/stains
 In acid fast staining, three different reagent or stains are used, they
are as follows,
1. Primary stain (Carbol fuchsin)
 Carbol fuchsin, a penolic stain soluble in lipoidal material of
mycobacterial cell wall, allows penetration and retention of red
stain
 Penetration is increased by application of heat, which drives carbol
fuchsin through lipoidal wall into cytoplasm
 All cells appear red in colour after application of primary stain

28. Acid fast staining continue….
27
Three reagent/stains
2. Decolourising agent (acid alcohol/ 20% H2So4)
 Smear is cooled before decolourisation, which allows waxy material
of cell substances to harden
 On application of decolourising agent, acid fast cells shows resistant
 Primary stain is more soluble in the cellular waxes than
decolourising agent, hence primary stain is retained by acid fast
bacteria (red) and not in case of non acid fast bacteria (colorless)

29. Acid fast staining continue….
28
Three reagent/stains
3. Counter stain
 Non acid fast bacteria absorbs counter stain and appears blue or
green colour e.g. Methylene blue, malachite green
 Acids fast bacteria retains red colour of primary stain

30. Acid fast staining continue….
29
Procedure
1. Prepare bacterial smear on clean and grease free slide, using sterile
technique.
2. Allow smear to air dry and then heat fix.
3. Cover the smear with carbol fuchsin stain.
4. Heat the stain until vapor just begins to rise about 60°C. Allow the
heated stain to remain on the slide for 5 minutes.
5. Wash off the stain with clean water.
6. Cover the smear with 3% v/v acid alcohol for 5 minutes or until the
smear is sufficiently decolourized, i.e. pale pink.
7. Wash well with clean water.

31. Acid fast staining continue….
30
Procedure
8. Cover the smear with malachite green stain for 1–2 minutes, using
the longer time when the smear is thin.
9. Wash off the stain with clean water.
10. Wipe the back of the slide clean, and place it in a draining rack for
the smear to air dry.
11. Examine the smear microscopically, using the 100 X oil immersion
objective.

32. Acid fast staining continue….
31
Fig. 5 Acid fast staining procedure

33. 9. Biochemical test (IMViC test)
32
 Living microorganisms are differentiated on the basis of various
enzyme-catalysed metabolic reactions
 Presence/absence of certain enzymes, intermeditary metabolites or
end products often give valuable information in identifying and
classifying of microorganisms
 Imvic test are used to diifferentiate Gram negative intestinal
bacteria (Family Enterobacteriacae) particularly E.coli,
Enterobacter cloacae and Enterobacter-Klebsiella group, on the
basis of their biochemical properties and enzymatic reactions in the
presence of specific substrate, It consist of four tests
1. I - Indole production test
2. M - Methyl red test
3. V - Voges - proskauer test
4. C- Citrate utilisation test

34. 1. Indole production test
33
 Media - Sulfide Indole Motility (SIM)
 Reagent - Kovac’s reagent (Consist of isoamyl alcohol, para-dimethyl
amino benzaldehyde and concentrated hydrochloric acid)
 Kovac’s reagent is used to determine the ability of the organism to
split indole from the amino acid tryptophan
 Bacterial species which possess enzyme tryptophanase, degrade
amino acid tryptophan to indole, pyruvic acid and ammonia

35. Indole production test continue….
34
 Indole production is detected by inoculating the test microorganism
into peptone water and incubating it at 37C for 48-96 hrs.
 Then add o.5 ml of Kovac’s reagent and mix gently
 A red colour in the alcohol layer indicates a positive reaction
(production of indole)
Fig. 6 Indole test

36. 2. Methyl red test
35
 Medium - MR-VP medium and it contains glucose, peptone and
buffer (buffer will neutralize weak acids so that only strong stable
acids will be detected by methyl red)
 It is used to identify bacteria to produce pyruvic acid from glucose
metabolism (fermentation of glucose)
 By production of acid, pH of medium falls and it is maintained
below 4.5
 Inoculate the test microorganism in MR-VP broth and incubate it at
37C for 2 to 5 days
 Then add five drops of 0.04% solution of methyl red, mix well and
result in the form of change in colour
 Red colour signifies positive while yellow signifies a negative test

37. Methyl red test continue…..
36
Fig. 6 Methyl red test

38. 3. Voges – proskauer (VP)test
37
 Medium - MR-VP broth
 Reagent - Barritt’s A and Barritt’s B which are mainly alpha naphthol
and potassium hydroxide respectively
 The test is mainly based on the principle of conversion of glucose to
acetyl methyl carbinol
 After break down of glucose it reacts with alpha naphthol and
potassium hydroxide to form a red colour solution, known as acetoin
 Inoculate the test microorganism in MR-VP broth and incubate it at
37C for 48 hrs.
 Then add 1 ml KOH and 3 ml of 5% solution of alpha naphthol in
absolute alcohol
 A positive reaction is indicated by the development of pink colour in
2 to 3 min.

39. Voges – proskauer (VP) test continue….
38
Fig. 7 VP test

40. 4. Citrate utilisation test
39
 Media - Simmon’s citrate media
 Reagent: Bromothymol blue indicator
 To determine the ability of an organism, using the enzyme citrase
and to use citrate as its sole carbon source for growth
 Principle –
1. Breakdown of ammonium salt gives ammonia and hydroxides.
These hydroxyl ions raise the pH and that changes the colour of
bromophenol blue indicator from green to royal blue
2. When the bacteria metabolize citrate, the ammonium salts are
broken down to ammonia, which increases alkalinity
3. The shift in pH turns the bromothymol blue indicator in the
medium from green to blue above pH 7.6.

41. Citrate utilisation test continue…
40
 Procedure
1. Inoculate the slant of Simmons citrate agar media (pH 6.9) with the
bacterial culture by streaking the surface of media.
2. Then incubate at 37°C for 1-2 days.
3. Observe and record the colour changes
 Observation
1. If the organism did not grow, the slant remains green. This
indicates test is negative, e.g. E. coli
2. If the organism grows, the slant became partially or completely
Prussian blue in colour, e.g. Klebsiella pneumonia

42. Citrate utilisation test continue…
41
Fig. 8 citrate utilisation test

43. 5. References
42
 Patil U, Kulkarni JS, Chaudhari AB, Chincholkar SB. Foundations
in microbiology. 4th ed. Pune: Nirali prakashan; 2006. Pg. no.
1.1-1.4, 5.1 – 5.2
 Kokare C. Pharmaceutical microbiology. 2nd ed. Pune: Nirali
prakashan; 2019. Pg. no. 1.1 -1.18
 https://en.wikipedia.org/wiki/Staining

44. Thank You
Maharashtra (India)