Common differential staining of bacteria

1. BACTERIAL STAIN
- Dr. ANKUR KUMAR

2. TOPICS for discussion-
• INTRODUCTION
• GRAM STAIN
• ACID FAST STAIN
• ALBERT STAIN

3. Introduction
• Structural details of bacteria cannot be seen under
light microscope due to lack of contrast.
• Hence, it is necessary to use staining methods to
produce color contrast and thereby increase the
visibility.
• Differential stain-
– two stains are used which impart different colors to
different bacteria or bacterial structures, which help in
differentiating bacteria.
• The most commonly employed differential stains are-
Gram stain- differentiates bacteria into gram positive and
gram-negative groups
Acid-fast stain- differentiates bacteria into acid fast and
non acid-fast groups
Albert stain- differentiates bacteria having metachromatic
granules from other bacteria that do not have.

4. • Principle :-
Some bacteria resist to decolourisation of primary
stain but others are easily decolourised and take a
secondary stain.
GRAM STAIN

5. GRAM STAIN
• Explanation-
 Gram stain describe morphology of bacteria and distinguishes
two categories of genera: the Gram positive which stain violet
colour and the Gram negative which stain light red colour.
 Gram positive have thicker and denser peptidoglycan layers in
their cell walls, which make them less permeable to the stain
than those of the Gram negative bacteria.
 Protoplasm of Gram positive bacteria is more acidic compare to
Gram negative bacteria, thus primary basic stain easily bind.
 The iodine has a critical role in enhancing this difference. The
violet dye and iodine combine to form an insoluble, dark purple
compound in the bacterial protoplasm and cell wall.
 This compound is dissociable in the decolourizer, which dissolve
and removes its two component from the cell. But the removal
is much slower from Gram positive than the Gram negative
bacteria, so that by correct timing the former stay dark purple
whilst the latter become colourless which take colour of counter
satin

6. Materials and Requirements
1. Violet Dye:
 Crystal Violet or methyl violet: 10gram
 Absolute Alcohol: 100ml
 Distilled water: 1litre
2. Grams iodine solution:
Iodine: 10g
Pottasium iodide: 20g
distilled water: 1 litre
3. Decolourizer:
Acetone/Absolute alcohol
4. Counter Stain:
Safranine (safranine 0.5% in distilled water)

7. Procedure
Take a clean, scratch free slide and make it grease free by moving over flame
Positive control & negative control smear made on same slide
If smear is made from colonies –
Take a drop of normal saline on slide
Heat the loop and allow it to cool. Then with the help of loop just touch the colony and spread it over the drop of
normal saline in circular fashion.
If smear is to be made from sample or broth suspension-
Then directly spread sample over the clean slide with the help of loop in circular fashion and no use of saline here
Dry the smear by waving it in air (air dried) & gently moving over the flame (heat fixed)
Smear covered with methyl violet for 1 minute.
Apply grams iodine for 1minute
Slide tilted to pour off grams iodine and washed under running water.
Smear is decolorised with acetone for 3 seconds (or with absolute alcohol for 30 sec.)
Then smears washed under running water immediately.
Apply safranin for 1 min and wash with water
Dry the slide by waving in air
Observe under 100x power of microscope after applying of oil

10. Interpretation
• Gram Positive – violet
• Gram negative – red
• Control :-
Positive control:- ATCC Staphylococcus aureus
Negative control:- ATCC Escherichia coli

11. ACID FAST STAIN/ Ziehl Neelsen stain
• Principle:
 Some bacteria resist to decolourisation with strong acid of
primary stain but others are easily decolourised and
doesn’t take the primary stain.
• Explanation-
Acid fastness (resist to decolourisation)- due
to presence of high content and variety of lipids,
fatty acids and higher alcohols- high molecular
weight hydroxy acid wax containing carboxyl
groups- called Mycolic acid

12. Materials and Requirements
• Zeihl-Neelsen carbol fuchsin
Basic fuchsin (powder) 5g
Phenol 25g
Absolute alcohol 50ml
Distilled water 500ml
• Sulphuric acid 25%
• Loeffler’s methylene blue 0.5%

13. STAINING METHOD
Take a clean, scratch free slide and make it grease free by moving over flame
The slide is put on a staining rack and covered with ZN carbol fuchsin
The slide is heated until steam rises but without boiling (intermittent heat) for 10
minutes.
Slide tilted to pour off the stain & washed in running water
Smear is decolourised with 20% sulphuric acid. The colour is changed to yellow-
brown.
After 1 min in the acid, the slide is washed and fresh acid poured on. This is
repeated several times until decolorization is complete (usually taking about 10
mins) finally, after washing the smear is only slightly pink.
The slide is now covered with Loeffler’s methylene blue for 2 mins.
Wash in running water
Dry and observe under oil immersion lens of the microscope

15. INTERPRETATION
• Acid fast organisms stain bright red
• other organisms, tissue cells and debris stain
blue.
• Quality control
 Positive : Mycobacterium species.
 Negative: A proven negative smear may be
used as the negative control

17. Albert Stain
• Principle:-
Basic dye stains volutine granule of diphtheria
strongly to dark bluish/ black colour but the
remaining bacteria takes the contrasting counter
stain (green colour).

18. Materials and Methods
• Primary stain- Albert Stain 1
 Toludine Blue 1.5 g
 Malachite Green 2 g
 Glacial acetic acid 10 ml
 Alcohol (95% ethanol) 20 ml
 Distilled water 1 lt
• Secondary Stain-Albert Stain 2
 Iodine 6 mg
 Potassium Iodide 9 mg
 Distilled water 1 lt

19. Procedure
Select and label a new unscratched slide.
Spread the specimen on the slide using a loop
Allow the slide to air dry for 15-30 minutes.
Fix the slide by passing it over a flame 3-5 times for 3-4 seconds each time.
Cover the slide with Albert stain 1 for 5 mins
Dry the slide with blotting paper
Cover the slide now with Albert stain 2 for 2 mins
Dry the slide with blotting paper
Now examine the slide under Oil Immersion Objective
Record the Results

21. • Interpretation
• KLB like green slender bacilli will be seen as
in their typical V-L arrangement

22. THANK
YOU