BACTERIAL STAINING

Dr. Harsha VarmaJMC,JhalawarMicrobiology Deptt.

Hans Christian Joachim Gram

• 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.

GRAM STAINING :•DANISH BACTERIOLOGIST HANS

CHRISTIAN GRAM (1880)•Based on this reaction, bacteria

classified into Gram positive and Gram negative bacteria.

•The cell wall compostion differences makes difference.

PRINCIPLE• 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. 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.

•A staining technique used to classify bacteria; bacteria are stained with gentian violet and then treated with Gram's solution; after being decolorized with alcohol and treated with safranine and washed in water, those that retain the gentian violet are Gram-positive and those that do not retain it are Gram-negative

Gram staining a Important Technique

Gram positive Bacteria• Gram positive

bacteria have a thick cell wall of peptidoglycan and other polymers. Peptidoglycan consists of interleaving filaments made up of alternating acetylmuramic acid and acetylglucosamine monomers. In Gram positive bacteria,between the cell wall and cell membrane, there is a "membrane teichoic acid".

Gram Negative Bacteria

• Gram negative bacteria have an outer membrane of phospholipids and bacterial Lipopolysaccharides outside of their thin peptidoglycan layer. The space between the outer membrane and the peptidoglycan layer is called the periplasmic space. The outer membrane protects Gram negative bacteria against penicillin and lysozymes.

Constituents of the Cell wall make difference

•Clean grease-free slide.•Bacteria tobe stained.•Inoculating loops- to transfer bacterial

suspension to slide.•Bunsen burner – to sterilise inoculating

loops before and after smear preparation.

•Pencil marker – to mark (particularly central portion of slide) where bacterial smear is applied

Basic requirements for staining:

•Smear preparation:•Putting of bacterial suspension

(bacteria in liquid) tobe stained on the central portion of slide in a circular fashion, air-dried, heat-fixed, the resultant preparation called bacterial smear- appears dull white.

Basic initial steps before staining:

1. Crystal violet – Primary stain2. Gram’s iodine- mordant/fixative3. Acetone (95%)- decoloriser4. Safranine/dilute carbol fuchsin –

counterstain

REQUIREMENTS – STAINING REAGENTS:

Organizing the Staining Bottles

1. Crystal violet - all bacteria take crystal violet- so all appears violet.

2. Iodine – Crystal Violet-iodine(CV-I) complex is formed.

3. Acetone- bacteria with high lipid content loose CV-I complex(appear colouless) but bacteria with less lipid content retains CV-I complex ( appear violet).

4. Safranine/ dilute carbol fuchsin – only colouless bacteria takes – appear pink.

•Crystal violet – 1 min - wash.•Iodine – 1 min – wash.•Acetone add drop by drop and watch out

colour comes out – wash immediately.•Safarnine/dilute carbol fuchsin – 1 min-

wash.•Allow to dry – examine under microscope.

Note: Results should be confirmed only with 100x.

PROCEDURE:

• SMEAR FIXATION:• 1) Heat fixation• a) Pass air-dried smears through a flame two or three times. Do not

overheat.

• b) Allow slide to cool before staining.

• 2) Methanol fixation• a) Place air-dried smears in a coplin jar with methanol for one minute.

Alternatively, flood smear with methanol for 1 minute.

• b) Drain slides and allow to dry before staining.

Method of Picking material from Agar platesWrong Right

Prefer to pick up colonies with loop and smear on Clean glass slide

•i. Relative amounts of PMN’s, mononuclear cells, and RBC's

•ii. Relative amounts of squamous epithelial cells, bacteria

•consistent with normal micro flora, which may indicate an improperly collected specimen

•iii. Location and arrangements of microorganisms

•Note any special character sticks

Observe the Following Under Oil Immersion lens

• Bacteria that manage to keep the original purple dye have only got a cell wall - they are called Gram positive.

• Bacteria that lose the original purple dye and can therefore take up the second red dye have got both a cell wall and a cell membrane - they are called Gram negative.

Colors makes the Difference in Gram staining

Colour:Purple colored bacteria – Gram positive Pink colored bacteria – Gram negativeShape:Spherical – cocciRod – bacilli Arrangement Cocci in clusters – staphylococciCocci in chains - streptococci

RESULT:

•If no microorganisms are seen in a smear of a clinical

•specimen, report “No microorganisms seen.”

• ii. If microorganisms are seen, report relative numbers and

•Describe morphology.• Observe predominant shapes of

microorganisms

Report as follows

GRAM POSITIVE COCCI IN CLUSTERS

GRAM POSITIVE COCCI IN CHAINS

GRAM NEGATIVE BACILLI

GRAM POSITIVE BACILLI

GRAM NEGATIVE COCCI

Gram stain (Mixed)

•Gram positive cocci in clusters:•1. Staphylococci species.•Gram negative cocci in chains:•1. Streptococci species.•Gram negative cocci:•1. Neisseria species.•Gram negative bacilli:•1. Escherichia coli•2. Klebsiella pneumoniae

EXAMPLES:

•Gram positive bacilli:

•1. Clostridium species.•2. Corynebacterium species.•3. Bacillus anthracis.

Gram-negative Pseudomonas aeruginosa bacteria (pink-rods).

30

(Bacillus anthracis), 31

Streptococcus pneumonia32

Streptococcus salivarius

Corynebacterium diptheria seen in Gram staining

Clostridium spp. seen in Gram Stain

Streptococcus pneumoniae in Sputum

Gram stain of Neisseria gonorrhoea,

Stains Several Fungi

Gram variable observations in Gram staining•The Gram staining procedure does not

always give clear-cut results. Some organisms are Gram-variable and may appear either Gram-negative or Gram-positive according to the conditions. With these types of organisms, Gram-positive and Gram-negative cells may be present within the same preparation

Overcoming in Gram Variable Observations•it is necessary that it is stained at two or

three different ages (very young cultures should be used). If an organism changes from positive to negative or vice versa during its growth cycle, this change should be recorded with a statement as to the age of the culture when the change was first observed. In case a Gram-variable reaction is observed it is also good to check the purity of the culture.

Why Gram Variability?•Some Gram-positive bacteria appear

Gram-negative when they have reached a certain age, varying from a few hours to a few days. On the other hand, some Gram-negative bacteria may become Gram-positive in older cultures. For this reason it is strongly recommended to use very young cultures for the staining procedure, after growth has become just visible.

Trouble shooting in Gram Staining method The method and techniques used.

Overheating during heat fixation, over decolourization with alcohol, and even too much washing with water between steps may result in gram-positive bacteria losing the crystal violet-iodine complex. The age of the culture. Cultures more than 24 hours old may lose their ability to retain the crystal violet-iodine complex.

The organism itself. Some gram-positive bacteria are more able to retain the crystal violet-iodine complex than others.

• By this method fairly good results may be obtained with very short staining

times, which are convenient when only one slide has to be stained.

• Flood the slide with crystal or methyl violet stain and allow to act for about 5

seconds.

• Tip off the stain and flood the tilted slide with iodine solution and allow to act

for about 5 seconds.

• Tip off the iodine and flood the tilted slide

• With acetone and allow this to act for only 2 seconds before washing it off

with water from the tap.

• Flood the slide with basic fuchsin counter stain and allow it to act for about 5

seconds. Wash off with water, blot and dry.

Quick GRAM METHOD

Modification in Gram staining methods

• Since the original procedure of Gram, many variations of the Gram staining technique have been published. Some of them have improved the method, others include some minor technical variants of no value.

• Bartholomew (1962) has pointed out that each variation in the Gram staining procedure has a definite limit to its acceptability. Any final result is the outcome of the interaction of all of the possible variables.

• All modified methods to be practised with caution should suit to the laboratory, and quality control checks.

Various modifications of Gram staining• 1. Kopeloff & Beerman’s Modification :

Primary stain is Methyl violet.

Decolourizer is Acetone/ Acetone-Alcohol mixture.

• 2. Jensen’s Modification :

Primary stain is Methyl violet.

Decolourizer is Absolute Alcohol.

Counter stain is Neutral Red.

• 3. Preston & Morrell’s Modification :

Primary stain is Crystal violet.

Decolourizer is Iodine-Acetone.

• 4. Weigert’s Modification:

Primary stain is Carbol Gentian violet.

Decolourizer is –Aniline-Xylol. Weigert stain is used to stain tissue sections.

Applications of Gram Staining• 1.Rapid presumptive diagnosis of diseases such as Bacterial meningitis.

• 2.Selection of Empirical antibiotics based on Gram stain finding.

• 3.Selection of suitable culture media based on Gram stain finding.

• 4.Screening of the quality of the clinical specimens such as sputum that

should contain many pus cells & few epithelial cells.

• 5.Counting of bacteria.

• 6.Appreciation of morphology & types of bacteria in clinical specimens.

THANK YOU