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027-1-MI

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1099X
MICROBIOLOGY
027-1-MI
BACTERIAL CLASSIFICATION
TABLE OF CONTENTS

PAGES
OBJECTIVES 2

RESOURCE MATERIAL 3

OVERVIEW 4
TAXONOMY 5
NOMENCLATURE 5
STRAINS 6
CLASSIFICATION USING BIOCHEMICAL TESTS 6
CLASSIFICATION USING GENETIC RELATEDNESS 9
CLASSIFICATION USING ANTIGENS 10

SELF-ASSESSMENT 11

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OBJECTIVES

The objectives indicate what you should know, understand and be prepared to explain upon completion
of this module. The self-assessment question will enable you to judge your understanding of the
maternal.

Upon completion of this module, the student should be able to:

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RESOURCE MATERIAL

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MICROBIOLOGY
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BACTERIAL CLASSIFICATION

OVERVIEW

One of the main features of a microbiology technologist’s work is the 1dentif1cation of bacteria so that
pathogens may be separated from insignificant organ1sms. Classification provides the framework or the
structure for identif1cation

Classification is merely the orderly arrangement of 1iving enties into groups. Bacterial classification is not as
simple as that of plants and animals since most bacteria are very uniform in Size and shape. The main groups,
as a result, must be very large, accommodating many bacterial species.

Bacterial classification traditionally is based on phenotypic properties such as:

 Stain1ng reactions
 Spore formation
 Presence of flagella
 Growth requirements
 B1iochemical patterns
 Genetic and immunological differences.

Bergey's Manual of Systematic Bacteriology comprises the most universally accepted bacteriological
classification scheme A summary of Bergey's classification is out1ined in Appendix B.

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TAXONOMY

Taxonomy is the Science of classification. Most living things are grouped in taxons or classification
groupings based on genetic relatedness of the group members to a common ancestor. The taxonomic groups
are.

 Kingdom
 Phylum (a group of related classes)
 Class (a group of related orders)
 Order (a group of related families)
 Family (a group of related genera)
 Genus (a group of related species)
 Species (the -lndividual" on Which all the other taxons are derived)

Bacteria are in the kingdom, Procaryotae (formerly Monera), Which is subdivided into -Sections-1n Bergey's
Manual. The only taxons used consistently are genus and species, with order and family empioyed less
frequently

Because bacteria have no easily discerible _-family tree-, slotting bacteria into a classical classification
scheme is artificial Bacterial species therefore, are defined primarily by their biochemical and physiological
attributes which change frequently As new data occurs their classification must also be adjusted accordingly.
As a result, some would say that bacterial taxonomy is the art rather than the science of classification.

NOMENCLATURE

Bacteria are named using latinized binomials which are a combination of a genus. name (analogous with our
surnames) followed by a species name (similar to a given name) Although the first letter of the genus name is
capitalized, the species name is always written in lower case. Because they are foreign words, bacterial names
are 1talicized Staphylococcus aureus or underlined taxonomic groups and Staphylococcus aureus Family
names are recognized by the end1ng "-aceae-, e.g. Neisseiraceae or Enterobactenaceae

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STRAINS

Bacterial strains are species variants. For example, three different patients may be infected with
Staphylococcus aureus but these three bacteria, although they are the same species, may differ slightly in
their colonial appearance or their ability to utilize various substrates. To indicate these slight differences, each
individual species isolated from a specimen is called a strain.

CLASSIFICATION USING BIOCHEMICAL TESTS

Most routine bacteriological identification is done using tests which detect the organism's ability to use
various carbohydrates or produce specific enzymes. Once these test values have been obtained they are
compared with diagnostic tables to determine identification.

Tables are constructed by collecting many strains of each particular species from a equally large number of
pat1ients

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Figure 1 illustrates a table where the percentage positives are recorded. (2 means 2% of the strains tested were
positive.)

In Figure 2, the percentages have been interpreted as follows:

 Values greater than 90 are considered positive


 Values less than 10 are considered negative
 Values between 10 and 90 are designated as variable reactions.

The patterns of positive and negative biochemical reactions comprise an organism’s biotype.

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Classification using large numbers of equally weighted values such as these is called numerical taxonomy.
Numerical taxonomy is the basis of the computer identification of bacteria as used in automated microbial
identification.

Numerical taxonomy can also be used to calculate the percentage of similarity between organisms. This can
graphically expressed as a dendrogram.

Depending on the bacteria being studied, a given level of percent similarity would be indicative of genus,
species or sub-species (strain) relatedness

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CLASSIFICATION USING GENETIC RELATEDNESS

These biochemical patterns comprise an organism’s phenotype. Biochemical reactions, unfortunately, glove
only an approximate reflection of an organisms’ underlying genetic structure or genotype. While lot is true
that each biochemical result iS often based upon the presence or absence of an enzyme which in turn is coded
for by a gene, the fact remains that a battery of 300 tests only assays 0.05-.20 (5-20%) of an organism's
genetic information. Therefore, although biochemical tests are useful for routine identification and species
separation, genetic studies provide the most definitive way of classifying bacteria.

There are several genetic parameters that may be used; the most common are the determination of guanine-
cytosine content and DNA hybridization studies

Because the guanine and cytosine percentage in an organism's DNA is constant for a species, the guanine +
cytosine (G+C) ratio may be used as a measure of genetic relatedness The G+C content in bactenal DNA
ranges from 0.25_.to 0.75 (25 to 75%) For example: -

Proteus/Providencia spp. have a range of 0.38 to 0 42 (38 to 42%), Escherichia spp from 0 50-0 53 (50-53%)
and Pseudomonas spp from 0.58 to 0.70 (58-70%). If the G+C range of two organisms is similar, they mayor
may not be members of the same species; however, if the ranges are very different the organisms cannot be in
the same species

If the DNA strands from two bacterial strains are denatured into single strands and then mixed together, they
will reaneal or rejoin (hybridize) almost completely if closely related but only partially if distantly related.
The degree of hybridization can then be measured (Further details are available in Appendix C)

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CLASSIFICATION USING ANTIGENS

The bacterial cell wall, the capsule, the slime layer and flagellar antigens are all used for identifying or typing
bacteria Although antigenic typing has limited usefulness for some genera, it may be extensively used for
others For example, the Escherichia and Salmonella genera have been divided into hundreds of types based
on their antigenic structure. These types are termed serotypes or serovar since the typing is done serologically.
The following table summarizes the most commonly used antigens.

As each new antigen is discovered, 1t is given a new number Many hundreds of bacteria may have a given O
or H antigen number descriptive of its serolog1cal uniqueness

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MICROBIOLOGY
027-l-MI
BACTERIAL CLASSIFICATION

SELF-ASSESSMENT

MARKS

[6] 1. What features are used for classifying bacteria?

[2] 2 Differentiate between biotype and serotype

[2] 3. How may genetic relatedness be determined?

[5] 4. a) describe the components of a bacterium's name.


b) write the genus and species name of a bacterium correctly.

[4] 5. Match the symbols in. Column A w1th the most appropriate descriptive statement in Column B

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MICROBIOLOGY
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BACTERIAL CLASSIFICATION
SELF-ASSESSMENT ANSWERS

1. -staining reactions
-spore formation
-presence of flagella
-growth requirements
-biochemical patterns
-genetic and immuno1ogical differences

2. biotype: species variation based on bichemical tests serotype: species variation based on antigenic
d1fferences

3. By determining the guanine and cytosine content or by the degree of hybridization

4. a} A bacterium' s name consists of a genus and species name.


b} The genus name beg1ns with an upper case letter and the species name starts w1th a lower
case letter; the name is underlined

5.

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APPENDIX C
DNA HYBRIDIZATION

When double-stranded bacterial DNA is heated, the DNA dissociates into single strands. These single strands
can reaneal via their hydrogen bonds upon slow coo1ing to reform the double-stranded DNA. If however,
single strands of DNA from two different bacterial species are combined and allowed to reaneal, the
percentage homology of the base sequences is a reflection of these organisms' genetic relatedness. When
double-stranded DNA is formed from two different species' DNA, this DNA is called a hybrid and the
process, DNA hybridization.

DNA hybridizat1ion-determined relatedness gives a genotypic picture not subject to variations caused by
mutations, plasmids, atypical biochemical reactions and variations

PROCEPURE (OVERVIEW)

Double-stranded DNA from bacter1um "A" is heated to denature into single-stranded DNA .

This single-stranded DNA from organism "A" is bound to sites on a cellulose fi1te are blocked

Unbound sites on the cellulose filler are blocked

Double “B” is grown in a medium containing a radioactive compound so that the radioactive substance are
incorporated into the bacterial DNA

These radioactively labeled fragments are allowed to incubate on the cellulose filter. Complementary bases
from the bacterium “B” will bind yto those of bacterium ”A”

After a prescribed time, unbound DNA is washed off and the amount of radioactive material left is used to
calculate the degree of hybridization.

Two bacteria are membrane of a species if their DNA relates sis 070(70) or greater, for example, Escherichia
coli and the Shigellas are greater than 070 related and should be in the same species. This technique is also
called a dot blot hybridization.

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MICROBIOLOGY
840-279
THEORY OF THE GRAM STAIN

THE MICHENER INSTITUTE for Applied Health Sciences

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MICROBIOLOGY
840-279
THEORY OF THE GRAM STAIN

ACKNOWLEDGEMENTS

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DMSION OF CONTINUING EDUCATION


840-279
THEORY OF THE GRAM STAIN

Table of Contents

PAGE

OBJECTIVES 1

RESOURCE MATERIAL 2

PURPOSE 3
Uses Of The Gram Staining In The Laboratory 3
Direct Staining Of Clinical Specimens 3
Prelimmary Identification Of Bacterial Growth 4

REAGENTS 4

OUTLINE OF GRAM STAIN PROCEDURE 5

THEORY OF THE GRAM STAIN 6


Primary Staining 7
Application Of Iodine (Mordant) 8
Decolourization 9
Secondary Staing (Counterstain) 12

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OBJECTIVES

The objectives indicate what you should know, understand and be prepared to explain upon completion
of this module. The self-assessment question will enable you to judge your understanding of the
maternal.

Upon completion of this module, the student should be able to:

1. Classify the Gram stain on the basis of the results obtained.

2. State the purposes of the Gram stain.

3. State how the results of a Gram stain may be of use m a diagnostic laboratory

4. List the Gram stain reagents.

5. Outline the Gram stain procedure.

6. State the bacterial cell feature responsible for the Gram reaction.

7 Explain the theory of the Gram reaction.

8. State the effect that each reagent has on bacteria when added m the correct sequence for the Gram
procedure.
9 State the consequence(s) of omitting any of these reagents.

10. Explain how the bacteria ill a Gram stained smear might be affected If the decolourizer is:
a) Left on for too long,
b) Not left on long enough.

11. State what the likely reaction of old is and/or damaged gram-positive bacteria.

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STAINING SERIES
840-279
THEORY OF THE GRAM STAIN

The cells of microorganisms are naturally transparent. Dyes or biological stains can be applied to slide
preparations of clinical material (patient specimens) or bacterial growth (a portion of a bacterial colony) to
render them Visible under the light microscope.

The Gram stain is the single most useful test m the clinical microbiology laboratory. The original Gram stain
was developed by the scientist, Gram, m 1884. Most microbiology laboratories use basically the same
procedure with a few modifications.

The Gram stain is an example of a differential stain; a staining procedure involving the application of two or
more dyes. In the Gram stain, cellular structures and microorganisms will take up either the blue colour of the
primary stain (crystal Violet) or the red colour of the secondary stain (safranin or carboI fuchsin).

The Gram stain is a differential stain also, because It separates bacteria into two large categories. Bacteria that
retain the crystal Violet dye and stain blue are referred to as gram-positive. Bacteria that take up the colour of
the safranin and appear pink to red are referred to as gram-negative.

PURPOSE

Uses Of The Gram Stain In The Laboratory

Direct staining of clinical specimens

Usually the first step m the laboratory analysis of clinical material from an infected body site is direct
microscopic examination. The microscopist can determine if there are gram-positive or gram-negative
bacteria in the specimen. Furthermore, cellular morphology and arrangement may provide clues to the
organism's identity. Direct examination provides important diagnostic information, which can guide antibiotic
therapy until culture results are available.

In some clinical situations the direct Gram stain is a STAT test. For example, when cerebral spinal fluid (CSF)
from a patient with symptoms of meningitis is submitted to the laboratory the Gram stain must be carried out
immediately. The choice of antibiotic used in the early stages of care may be altered based upon the reporting
of gram-positive versus gram-negative bacteria in the CSF.

Gram staining can also be used to differentiate and quantitate epithelial and inflammatory cells (segmented
neutrophils), thus providing information concerning the state of infection and the quality of the specimen. For
example, the presence of many neutrophils suggests the presence of

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a disease process or an inflammatory response. Excessive epithelial cells without neutrophils, however, in
most cases suggest that either a poor sample was collected or there was little evidence of infection at time of
collection.

Gram staining information is helpful when setting up specimens for culture. For example, the presence of
large numbers of gram-positive organisms and gram negative rods in a direct stain of a clinical specimen
might suggest the need for the addition of selective culture media to assist m the separation of pathogens in
culture.

Preliminary Identification of Isolated Bacterial

The Gram strain is often the first test performed on bacterial growth in culture. The Gram reaction of the
organism in question is essential information, winch assists in the choice of the correct Identification protocol.
Inexperienced technologists may be tempted to omit this first step resulting In misidentifications and patient
mismanagement.

REAGENTS

Crystal Violet: Primary Stain


This dye is blue-Violet m colour.

Iodine Solution: Mordant


Some authors refer to tins as a "trapping agent"

Acetone/Alcohol: Decolorizer
Either acetone of ethyl alcohol can be used independently as a decolorizer.

Safranin: Counterstain or Secondary Stain


Dilute carbol fuchsin is sometimes used instead of safarnin.

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OUTLINE OF THE GRAM STAIN PROCEDURE

A number of Gram staining methods exist; all of which are modifications of the original Gram method (e.g.
Hucker's and Atkan's stains). For a detailed account of the Hucker's method, refer to the Supplement at the
end of this section. Although the formulation of reagents and the staining times vary from method to method,
the basic procedure is as follows:

Preparation of the smear· Transfer clinical material (i.e. an emulsion of tissue, material from a swab, a
concentrate of body fluid) to a sterile glass microscope slide; ensuring that the preparation is not too thick. A
section of a bacterial colony can be removed with a sterile wire and emulsified m a loopful of saline. The
emulsification m saline should be done gently to maintain the natura1 arrangement of cells, such as chains
and clusters. Ideally, smears should be allowed to air-dry

Fixing the preparation: Fixing the smear prevents the preparation from washing off the slide and kills most
of the organisms. Smears can be fixed by passing them through (or over) a source of heat (e.g. a Burnsen
burner or incinerator). Caution -excessive heat can cause distortion of organisms. Alternatively, smears can be
fixed by immersion in 95% methanol for 2 minutes.

Primary staining: Flood the smear with the crystal Violet solution and let it stand for a specified time
(usually 1-2 minutes depending upon the method). The stain is washed off the slides with tap water. Slides are
then drained to remove excess water.

Mordant: Flood smears with an Iodine solution and let it stand for a specified time (usually 10 seconds to 1
minute depending upon the method). The Iodine solution is washed off the slides with tap water. Slides are
drained to remove excess water.

Decolourlzation: Flood smears with an ethanol-acetone solution or 95% ethanol. Allow decolorization to
proceed until no further colour (the blue dye) flows from the smears. This step usually takes from 10 to 20
seconds, depending upon the thickness of the preparation and the type if decolorizer used. The slowest agent
is 95% ethanol. An intermediate-acting agent is 50:50 mixture of 95% ethanol and acetone. The fastest agent
is pure acetone. Decolourzation is halted by washing slides in tap water. Slides are drained to remove excess
water. The I1mmg of this step is critical. Over-decolourization results in "true" gram positive bacteria staining
as gram-negative bacteria. Likewise under-decolourization results in "true" gram-negative bacteria staining as
gram-positives.

Secondary staining: Flood smears with either a safranin or carbol fuchsin solution and let it stand for a
specified time (usually 30 seconds to one minute depending upon the method). The stain is washed of the
slides with water. Slides are drained to remove excess water. Allow slides to air-dry before examination under
the microscope.

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THEORY OF THE GRAM STAIN

The proportions of lipid (lipoprotein and lippolysaccharide) and peptidoglycan (also called mucopeptide) in
the cell wall of gram-negative and gram-positive bacteria are thought to be responsible for their different
Gram reactions (Table I and Figure I).

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Primary Staining

Basic stains are used m the Gram stain procedure. These stains are positively charged, forming ionic bonds
with negatively charged groups.

When crystal violet, a basic stain, contacts a bacterium, the stain diffuses through cell wall pores bonding
with negatively charged groups within the cell (e.g. phosphate groups m nucleic acids). Since these negatively
charged groups are distributed throughout the bacterial cell, the entire cell stains uniformly blue (Figure 2).

Washing briefly with water removes excess stain but does not decolorize the bacteria.

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Application of Iodine (Mordant)

When Iodine IS added (Figure 3), it diffuses into the cell combining With the already bound crystal violet to
form a large molecular complex: the crystal Violet-Iodine complex (CVI).

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The Iodine, by forming a larger complex With the crystal Violet, traps the crystal Violet by making It more
difficult for the crystal violet to pass through the cell wall pores than If It was uncomplexed. Thus the Iodine
is often referred to as a "trapping agent"

This complex is stable m water but is soluble to varying extents In solvents such as acetone and alcohol, both
of winch may be used either individually or m combination for the next step decolourizatlon.

Decolourization

Decolourization is the most important step of the Gram stain procedure. Timing is critical. Ethanol (95%),
acetone, or a mixture of equal volumes of acetone and (95%) ethanol are commonly used.

The alcohol concentration is Important. Indications are that 50-75% aqueous alcohol decolorizes the cell more
rapidly than higher concentrations. For tins reason, all water should be drained from the slide before applying
the decolourizer.

Whichever decolourizer is used, It seems that the following events occur Simultaneously (Figure 4).

Lipid is extracted; the loss of lipid creates larger pores in the cell wall. Verious elements now pass much
easier into and out of the more permeable cell wall.

Peptidoglycan is dehydrated thus reducing pore Size and Inhibiting movement of elements Into and out of the
cell.

CVI complex is slowly dissolved.

Gram-negative bacteria have a high ratio of lipid to peptidoglycan In their cell wall. The loss of lipid during
decolourization Increases cell wall pore Size and permits CVI complexes Inside the cell to escape to the
outside.

Gram-positive bacteria have a high ratio of peptidoglycan to lipid In theIr cell wall. During decolourization
the cell wall pore Size shrinks as peptidoglycan shrinks. The CVI complexes inside the cell tend not to escape
to the outside as readily as In grant-negative bacteria.

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Gram-negative bacteria will appear colourless microscopically after only a few seconds of decolourization
(Figure 5).

After a given tune


(long enough for complete decolourization of gram-negative cells), gram positive cells will retain the CVI
complex and appear bluish-purple (Figure 6).

If decolourization time is too short, differentiation may not occur. Thus all bacteria, even those which are
characteristically gram-negative, may appear blue. This ix called under decolorinzation.

Conversely, If the decolourizatlon tune is too long, all organisms (including organisms which are normally
gram-positive), may appear red. This IS called over-decolourization.

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Secondary Staining (Counterstain)

When safranin or an appropriate substitute (e.g. dilute carbol fuchsin) ix applied to the decolourized slide, It
has no effect on gram-positive cells (Figure 7). The binding sites are blocked with the primary stain.

However, since CVI


binding sites In gram-negative cells have been freed by the decolourizer, they now form bonds With the red
counterstain (Figure 8).

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After counterstaing, gram-positive cells remain blue but gram-negative cells staIn red.

The integrity of the cell walls of the bacteria being stained can determine the final Gram reaction. Cells that
are sampled from a bacterial colony that is 48 or more hours old often have a cell wall that is more porous.
This phenomenon can result ill a gram-positive bacterium appearing gram-negative or pink due to the loss of
the primary stain and the uptake of the counterstain. Cells exposed to disinfectants/detergents/antibiotics may
also stain atypically (as above) due to cell wall damage.

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STAINING SERIES SUPPLEMENT


840-279-1
GRAM STAIN METHODOLOGY
(HUCKER'S MODIFICATION)

MATERIALS:

CRYSTAL VIOLET

Solution A

Crystal Vtolet (0.90 (90%) dye content) 2 gm


Ethyl alcohol 0.95 (95%) 20 mL

Solution B

Ammonium Oxalate 0.8 gm


Distilled Water 80.0 mL

Mix solutions A and B Store for 24 hours then filter before use.

Gram's Iodine:

Iodine 1 gm
Potassium iodide 2 gm
Distllied water 300 mL

NOTE The iodine will go ito solution more readily If the iodine and potassium iodide are first
dissolved in 5 mL of the water Then the remainder of the water can be added. Store in an amber glass bottle

Decolorizer Acetone-Alcohol

95% Ethyl Alcohol 1 part


Acetone 1 part

Counterstain: Safranin (or dilute carbol fuchsin)

1 Safranin
Safranin 0 (0.025 (2 5%) solution in
0.95 (95%) ethyl alcohol) 10 mL Distilled water 90 mL

OR

2 Dllute Carbol Fuchsin


Ziehl-Neelsen's Carbol Fuchsin 1 part
Distllied Water 19 parts

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METHOD: Gram Stain Procedure

1. Flood smear with crystal violet and allow to stand for approximately 1 minute.

2 Rinse with tap water.

3. Rinse with Gram's iodine (or shake excess water off each shde) .

4. Flood with Gram's iodine and allow to stand for approximately 1 minute.

5. Gently rinse the smear with running tap water Shake off excess to prevent dilution of the acetone-
alcohol
6 Decolourize with acetone/alcohol for approximately 3-4 seconds.

NOTE This is the cristical step. The time of decolourization is variable depending upon many factors such as,
thickness of the smear, type or organism, etc. An adequate decolourization time will come with
practice

7 GentlY rinse with tap water, shake off excess

8. Rinse slide with safranin, flood with counterstain and let stand for approximately 1 minute. (Use
safranin for routine use, carbol fuchsin is recommended for staining specimens potentially containing
anaerobes.)

9 Rinse off with running tapwater. Drain.

10 Air dry

11 Wipe back of smear with tissue to remove stain residue

12. Examine with oil immersion objective.

RESULTS.

Gram-positive organisms stain purple


(Control organism. Staphylococcus species)
Gram-negative organisms stain red or pink
(Control organism Niesseria species)

NOTES

 Gram stains of bacteria will not always give typical morphology if the organism was grown on
selective media..
 Crystal violet must be filtered prior to dispensing into squeeze bottles.
 To remove oil for slide storage, dip slides several times into a coplin Jar containing xylol. Ar dry.

REFERENCE:

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