BACTERIAL STAINING
TECHNIQUES
STAINING
• Staining is a biochemical process usually conducted to increase the contrast of a
microscopic image.
• It is used to differentiate the stained portion of the image from the rest.
• The purpose of staining might also differ as some staining procedures are used to
1) differentiate between two groups of microorganisms (Gram Staining)
2) while some are used to observe motility and presence of a capsule.
• Staining is a part of biochemistry where a class-specific dye is added to a
substrate to qualify or quantify the presence of a certain compound.
• Staining, however, is not limited to biological samples, as it can be used to study
the lamellar structure of polymers and the structure of block compounds.
• Bright field microscopes are commonly used to observe
stained cells or other biological materials
• It is also difficult to observe colorless substances under a
microscope, which is why the concept of staining is so
popular.
• Microscopic techniques in hematology, histopathology,
and clinical microbiology use staining as a method to
study and diagnose diseases at the microscopic level.
• In some cases, staining is unnecessary, for example when
microorganisms are very large
• In such cases, a drop of the microorganisms can be placed
directly on the slide.
• Cover the specimen with coverslip and observe.
• A preparation such as this is called a wet mount.
• A wet mount can also be prepared by placing a drop of culture on a cover‐slip (a glass
cover for a slide) and then inverting it over a hollowed‐out slide.
• This procedure is called the hanging drop.
• The wet mount tend to dry out quickly
under the heat of the microscope light;
it is simpler to perform than the wet mount,
but it is useful for short-term observation
only.
• The hanging drop is a more complex technique,
but it allows for longer-term observation and more
reliable observation of motility.
TYPES OF STAINING
• Staining is of different types depending on their purpose
1) Simple staining using a single stain to provide a contrast between the sample
and the background;
• Staining can be performed with basic dyes such as crystal violet or
methylene blue, positively charged dyes that are attracted to the
negatively charged materials of the microbial cytoplasm.
• Such a procedure is the simple stain procedure.
• An alternative is to use a dye such as nigrosin or Congo red, acidic, negatively charged dyes.
• They are repelled by the negatively charged cytoplasm and gather
around the cells, leaving the cells clear and unstained.
• This technique is called the negative stain technique.
• The main purpose of Negative staining is to study the morphological
shape, size and arrangement of the bacteria cells that is difficult to stain.
eg: Spirilla
PROCEDURE
1. Clean glass slide was taken and was washed and dried.
2. With the help of inoculation loop, a drop of the bacterial suspension is transferred at the
centre of the slide.
3. The drop is spreads over the centre of the slide to about 1 cm diameter and allow to air
dry.
4. The bacterial smear is fixed by passing the slide rapidly over the flame
5. The slide was kept on the staining tray and 5 drops of stain was added for a
designed period.
6. The extra stain was poured off and the smear was washed gently under slow running tap
water.
7. The slide was then blot dried using blotting paper.
8. The slide was then examined under oil immersion objective.
2) Differential staining using more than one dyes in order to
differentiate between cells or between organelles of a cell.
• The differential stain technique distinguishes two kinds of organisms.
• An example is the Gram stain technique.
• This differential technique separates bacteria into two groups, Gram‐
positive bacteria and Gram‐negative bacteria.
Other staining techniques include endospore staining for endospores, acid-
fast staining for fastidious organisms etc
Gram Staining
• The Gram stain was developed by Christian Gram in 1884
• This test differentiates the bacteria into Gram-Positive and Gram-
Negative Bacteria.
• Gram-Positive: Streptococcus, Staphylococcus, Corynebacterium,
Listeria, Bacillus, Clostridium, etc.
Gram-Negative: E. coli, Salmonella typhi, Shigella spp,
Pseudomonas, Neisseria gonorrhoeae etc.
Gram Stain Reagents
1) Crystal violet
Solution A
Crystal violet – 2g
Ethanol (95%) – 2ml
Solution B
Ammonium oxalate – 0.8g
Distilled water -100mL
Solution A and Solution B are mixed 24 hours before use
2) Grams iodine
Iodine – 1g
Potassium iodide – 0.3g
Methanol/ethanol – 45ml
3) Decolorizer - 95% alcohol
4) Counterstain
Saffranin – 2.5g
Ethanol – 100ml
Dilute 100ml of the stain with 100ml of distilled water
Principle of Gram Stain
• The two major groups of bacteria can be divided into gram-positive and gram-negative.
• The Gram stain technique is based on the differential structure of the cellular membranes and cell
walls of the two groups.
• Gram-positive organisms contain a highly cross-linked layer of peptidoglycan that retains the
primary dye, crystal violet (CV), following the application of the mordant, iodine (I).
• The iodine and crystal violet form a complex (CV-I) within the peptidoglycan.
• When decolorizer is applied to the cells, the CV-I complex remains within the cell, making it
appear dark purple to blue.
• The gram-negative organisms do not contain a thick cross-linked layer of peptidoglycan.
• The peptidoglycan is loosely distributed between the inner cell and the outer cell membranes.
• Following the application of the crystal violet and iodine, the CV-I complexes are not trapped
within the peptidoglycan.
• Application of the decolorizer dehydrates the outer cellular membrane, leaving holes in the
membrane and effectively washing or removing the CV-I complex from the cells.
• The cells appear colorless.
• To make the colorless cells visible, a secondary stain, saffranin, is applied, leaving the gram-
negative cells pink.
Procedure of Gram Stain
1. The glass slide is cleaned with distilled water and dried with tissue paper.
2. With the help of inoculation loop, a drop of the bacterial suspension is transferred at the
center of the slide.
3. The drop is spreads over the center of the slide to about 1 cm diameter and allow to air dry.
4. The smear is fixed by passing the slide rapidly over the flame.
5. The slide is placed on the staining tray and the smear is flooded with crystal violet and kept
for one minute.
6. Then the slide is washed with the tap water to remove excess stain.
7. The smear is flooded with Gram’s iodine and kept for one minute.
8. Then the slide is washed with the tap water to remove excess stain.
9. The smear is decolorized by adding drops of 95% ethanol.
10. Then the slide is washed with water to stop the decolourization procedure.
11. The smear is flooded with saffranin for 45 seconds and rinsed with water.
12. The stained slide is air dried and observed under microscope
Result Interpretation of Gram Stain
• Gram-positive: Blue/Purple Color
• Gram-Negative: Red/Pink Color
