1. Place slide with heat fixed smear on staining tray.

2. Gently flood smear with crystal violet and let stand for 1 minute.

3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.

4. Gently flood the smear with Gram’s iodine and let stand for 1 minute.

5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
The smear will appear as a purple circle on the slide.

6. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol
drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to
over-decolorize.

7. Immediately rinse with water.

8. Gently flood with carbol fuchsin / safranin to counter-stain and let stand for 45 seconds.

9. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.

10. Blot dry the slide with bibulous paper.

11. View the smear using a light-microscope under oil-immersion.

 Principle:
The most widely used staining procedure in microbiology is the Gram stain, discovered by the Danish
scientist and physician Hans Christian Joachim Gram in 1884. Gram staining is a differential staining
technique that differentiates bacteria into two groups: gram-positives and gram-negatives. The
procedure is based on the ability of microorganisms to retain color of the stains used during the gram
stain reaction. Gram-negative bacteria are decolorized by the alcohol, losing the color of the primary
stain, purple. Gram-positive bacteria are not decolorized by alcohol and will remain as purple.
After decolorization step, a counterstain is used to impart a pink color to the decolorized gram-negative
organisms.
1. Gram positive bacteria: Stain dark purple due to retaining the primary dye called Crystal Violet
in the cell wall [made up of thick layer of peptidoglycan (50-90%) & Lipoteichoic acid (LTA)].
Example: Staphylococcus aureus
2. Gram negative bacteria: Stain red or pink due to retaining the counter staining dye called Carbol
fuchsin / safranin (thinner layer of peptidoglycan (10% of the cell wall), lose the crystal violet-iodine
complex during decolorization with the alcohol), (contain additional lipid layer).
Example: Escherichia coli
Coccus (pleural: Cocci):
Spherical bacteria; may occur in pairs (diplococci), in groups of four (tetracocci), in grape-like clusters
(Staphylococci), in chains (Streptococci) or in cubical arrangements of eight or more (sarcinae).
For example: Staphylococcus aureus, Streptococcus pyogenes
Bacillus (pleural: Bacilli):
Rod-shaped bacteria; generally occur singly, but may occasionally be found in pairs (diplo-bacilli) or
chains (streptobacilli).
For example: Bacillus cereus, Clostridium tetani
Spirillum (pleural: Spirilla):
Spiral-shaped bacteria
For example: Spirillum, Vibrio, Spirochete species.

Typical Gram-negative bacteria:

1. Bordetella pertusis, the causative agent of whooping cough
2. Salmonella typhi, the causative agent of typhoid
3. Vibrio cholera, the causative agent of cholera
4. Escherichia coli, the normally benign, ubiquitous, gut-dwelling bacteria

Typical Gram-positive bacteria:

1. Staphylococci such as Staphylococcus epidermidis and Staphylococcus aureus which is a common
cause of boils.
2. Streptococci such as the many species of oral streptococci, Streptococcus pyogenes which causes
many a sore throat and scarlet fever and Streptococcus pneumoniae which causes lobar pneumonia.
3. Clostridia such as Clostridium tetani, the causative agent of tetanus (lockjaw).
4. Actinomyces such as Actinomyces odontolyticus which is found in mouth.
5. Species of the genus Bacillus such as Bacillus subtilis which are common microbes living in soil.
Generally cocci are Gram-positive but there are exceptions. The most significant from a clinical point of
view is the gonococcus, Neisseria gonorrhoea which typically appears as a Gram-negative diplococcus
looking very much like a pair of kidney bean.
PRINCIPLE
Organisms such as Mycobacteria are extremely difficult to stain by ordinary methods like Gram
Stain because of the high lipid content of the cell wall. The phenolic compound carbol fuchsinis
used as the primary stain because it is lipid soluble and penetrates the waxy cell wall. Staining by
carbol fuchsin is further enhanced by steam heating the preparation to melt the wax and allow the
stain to move into the cell. Acid is used to decolorize nonacid-fast cells; acid-fast cells resist this
decolorization. The ability of the bacteria to resist decolorization with acid confers acid -fastness to
the bacterium. Following decolorization, the smear is counterstained with malachite
green or methylene blue which stains the background material, providing a contrast colour against
which the red AFB can be seen.

Acid alcohol can also be used as decolorizing solution, resistant organisms are referred to as Acid
Fast Bacilli (AFB) or Acid Alcohol Fast Bacilli (AAFB).

Among the Mycobacterium species, M. tuberculosis and M. ulcerans are strongly acid fast,
whereas M. leprae is weakly acid fast.

RESULTS AND INTERPRETATION

 Acid Fast Bacilli : Red, straight or slightly curved rods, occurring singly or in small
groups, may appear beaded
 Cells : Green (malachite green) or Blue (methylene blue)
 Background material : Green (malachite green) or Blue (methylene blue)

List of Acid Fast organisms

 Mycobacterium spp: Acid Fast
 Cyst of Cryptosporidium: Acid Fast
 Cyst of Isospora: Acid Fast
 Nocardia spp: Partial Acid Fast
 Rhodococcus spp: Partial Acid Fast
 Legionella micdadei: Partially acid fast in tissue

SAMPLES - sputum, fluids, tissue, and urine, bronchial washing & pleural fluid.

SPUTUM DISPOSAL –
At home : patients should add a little water to the sputum collected, boil it for 20 minutes, and
dispose of it in running water.
At hospital : mugs with zip pouches attached to them to collect patient's sputum. The
pouches are disposed of in high-pressure autoclaves.
 FLUORIDE ESTIMATION

The procedure depends on the formation of unionized zirconium fluoride salt thus
decreasing the color of zirconium alizarin.

Reagent: - 1. Acid Zirconium alizarine reagent

Apparatus: - A. Nessler’s tube 100 ml

B. Volumetric pipette 5 ml.

Technique: - 1. Place in clear clean Nessler’s tube 100 ml of sample to be tested.

2. Pipette exactly 5 ml of reagent acid zirconium alizarine.
3. Allow to stand for about one hour.
4. Shortly before one hour fill one of the 250ml tube with treated sample and
place in the middle compartment of the base. Fill two more tubes with
untreated sample and place in outside compartments of the base.
5. Compare the sample with standard by sliding the slide to the left or right
as required.

Calculation: - The value of colour so matches as against the value on the slide represents
ppm of fluride.
Fluoride ions have dual significant in water supplies. High concentration of F- causes
dental fluorosis (disfigurement of the teeth). At the same time, a concentration less than
0.8mg/L results in ‘dental caries’.

Hence, it is essential to maintain the F- concentration between 0.8 to 1.0mg/L in

drinking water.

Fluoride (F-) reacts with zirconium, dissociating a portion of it into a colourless complex
anion (Zr F6-). As the amount of fluoride increases, the colour produced becomes
progressively lighter.
ESTIMATION OF FAT CONTENT

GERBER'S METHOD:
The protein is first Precipitated and then dissolved by the
acid ,the fat globules are thus freed . the amyl alcohol produces a greater
difference in surface tension between the fat globules and the liquid . thus
causing an aggregation and separation of the fat .
Reagents :- 1. Sulphuric acid of specific gravity 1.82 to 1.83
2. Amyl alcohol
Technique:-
1.Pipette of 10ml of reagent sulphuric acid into Gerber’s tube
2. Add 11 ml of milk sample carefully avoiding mixing
3. Add 1ml of reagent Amyl alcohol similarly
Note:- If these have been carefully and correctly added . three layers
should now be present in the tube.
4. Cork the tube and mix the three layers.
Add little amount of distilled water to cover the empty space up to
the neck of tube in cases the Gerber’s tube is of larger capacity.
transfer the tube to the centrifuge machine and rotated for four
minutes.
5. Read off the fat layer from the bottom of the meniscus adjusting the
lower ends of the fat layer against a scale reading by rotating the
cork upwards or downwards
6. this gives the percentage of fat content directly in a given sample of
milk.

Result :- 3.4% Fat (Chitale Milk)

OTHER METHOD – STOKE’S METHOD
ESTIMATION OF NITRITES (NO2) IN WATER
Objective – Presence of NitrIte represent second stage of proteolysis including
sewage, or organic pollution.
(1) Starch Iodine test – 10 cc of sample water is taken in a test – tube. 3 drops
of potassium iodide is added 1 cc dilute Sulphuric Acid and 1 cc starch are
added. Appearance of blue colour indicates presence of NitrIte.

(2) Ilosvay’s Test – 50 cc sample water is taken in Nessler’s tube 1 cc Ilosvay ‘A’
(Naphthylamine in acetic acid) and 1 cc Ilosvay ‘B’ (Sulphuric Acid) are
added. Allowed for 15 minutes. Appearance of pink indicates presence of
Nitrites.

ESTIMATION OF NITRATES (NO3)IN WATER.

Objective – Nitrates are stable salts derived from organic pollution or from
geological sources. It indicates past pollution.
Qualitative Test:
(i) Brucine Test – 10 cc sample of water is taken in a test. Tube few drops of
Brucine reagent is added. 2 cc of concentrated H2So4 (Sulphuric Acid) is
added along the side of the test – tube development of pink at the junction
of two layers indicate the presence of No3 in water.

(ii) Diphenylamine Test – 5 cc sample of water is taken in a test tube. Three to

four drops of reagent is added (Diphenylamine). 2 cc of concentrated H2So4
is added along the sides of the test tube. The gradual appearance of blue
colour indicates the presence of No3 in water.