gram staining guest lecture jipmer pondcherry

8/3/2019 Gram Staining guest Lecture JIPMER Pondcherry

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GramStaining inClinical Microbiology

Dr.T.V.Rao. MD

Professor of Microbiology

Travancore Medical College, Kollam. Kerala

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The Guest Lecture presented by Dr.T.V.Rao MD

at

Tenth National Workshop on

Simple Diagnostic Methods in ClinicalMicrobiology

29th November to 3rd December 2011

Department of Microbiology

Jawaharlal Institute of Postgraduate MedicalEducation & Research, Pondicherry

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Working at Mansa General Hospital Mansa

Republic of ZambiaTaught many lessons, to understand Infection and Ignorance

are important causes of Morbidity and Mortality

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Hans Christian Gram The Gram stain was

devised by the Danish

physician, Hans Christian

Gram, while working in

Berlin in 1883. He laterpublished this procedure

in 1884. At the time,

Dr. Gram was studying

lung tissue sections frompatients who had died of

pneumonia.

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First Paper on Gram Staining

In his paper, Dr. Gram described how he wasable to visualize what we now callStaphylococcus, Streptococcus, Bacillus, and

Clostridiain various histological sections.Interestingly, Dr. Gram did not actually usesafranin as a counter stain in the originalprocedure (Gram negative cells would be

colorless). He instead recommended usingBismarck brown as a counter stain to enabletissue cell nuclei to be visualized.

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Carl Weigert (1845-1904)

German

pathologist Carl

Weigert (1845-

1904) from

Frankfurt, added

a final step ofstaining with

safranin.

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Traditional Definition of Gram stain

A method of staining bacteria using a violetstain. The gram staining characteristics(denoted as positive or negative). A heat fixed

bacterial smear is stained with crystal violet(methyl violet), treated with 3%iodine/potassium iodide solution, washedwith alcohol and counterstained. The method

differentiates bacteria into two main classes,gram-positive and gram-negative.

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The Cell walls differ

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Gram Positive should not be Mistaken

In the Gram Stain technique, two

positively charged dyes are used:

crystal violet and safranin. The useof the designation gram-positive

should not be confused with the

concept of staining cells with asimple stain that has a positive

charge. Dr.T.V.Rao MD 9


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Gram staining observation

Basic Principle in Kochs postulations

The first of

Kochs postulate

that thesuspected the

organism should

always be found

in association

with the disease.Dr.T.V.Rao MD 10


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Poor quality of slides

Can be corrected

Use of glass slides

that have not

been pre cleanedor degreased ?NOTE: Storing slides ina jar with 95% ethanol

will ensure clean slides.

Drain excess alcohol or

flame slide before use.

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Four Major Steps in Gram Staining

There are four basic steps of the Gram

stain, which include applying a primary

stain (crystal violet)or Methyl violet to aheat-fixed smear of a bacterial culture,

followed by the addition of a mordant

(Gram's iodine), rapid decolorization withalcohol or acetone, and counterstaining

with Safranin or basic fuchsin.

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Organizing the Staining Bottles

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Making a Smear

First prepare your

slide. You do this

by placing bacteriaon a slide in a drop

of water, allowing

them to dry andthen heat fixing

them. Heating

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Correct preparation

Smear preparation: Proper smear

preparation should produce a monolayer of

organisms sufficiently dense for easy

visualization but thin enough to reveal

characteristic morphological characteristics.

Use clean, new glass slides.

NOTE: When using the same pipette or swab,always inoculate culture media first

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Method of smearing the MaterialWrong Right

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Using Methanol is it Better than

Heat Fixation ?

Fix the smear with95% Methanol

Which will help in

prevention ofdistortion of cells

Helpful in

Microscopicobservation ofCSFand Urine

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Making Multiple smears in same slide

conserve resources

Making multiple

smears make the

optimal use of theslide.

Reduces the

economic costsand saves the

technical time.

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Steps in Gram Staining Procedure-

Follow the Clock

1On a rack, flood with filtered crystal violet( Methyl violet ) 10 sec2 Wash briefly in water to remove excess crystalviolet

3. Flood with Grams iodine 10 sec 4. Wash briefly in water, do not let the section

dry out. 5. Decolourise with acetone for few seconds


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Proceed in organized Fashion

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Step 1

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Step 2

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Step 3

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Step 4

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Step 5

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How long you keep Iodine in the

Laboratory ???

The Grams Iodine we make in

the laboratory from basic

chemicals

How long we can use it ?

Why we have to make frequently ?

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Most Critical Step in Gram staining

The most critical

step of gram staining

is the decolorization

step as crystal violetstain will be

removed from both

G+ve & G-ve cells ifthe decolorizing

agent(e.g alcohol ) is

left on too long.Dr.T.V.Rao MD 27


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Acetone used with Caution

Acetone is a morerapid decolorizesthan alcohol and

must be used withsome care.

Excessivedecolorization turns

Gram positiveappear as Gramnegative

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Which alcohol is better

Several alcohols have been studied, and

it has been reported that the more

complex the alcohol, the slower the

decolorization action. As the carbon

chain lengthens, decolorization is slower.

Conn found in practice, however, noknown advantage can be gained by

substituting the higher alcohols for ethyl

alcohol.Dr.T.V.Rao MD 29


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Step 6

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Which counterstain is better

Some bacteria whichare poorly stained by

Safranin, suchas Hemophilus

spp., Legionella spp., and some anaerobicbacteria, are readily

stained by basicfuchsin, but notSafranin

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Step 7

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Caring the stained slide

After the counterstain hasbeen rinsed off, the slideis placed between someabsorbent paper and the

excess water gentlyblotted off.Care must be taken notto rub the slide with the

blotting paper becausethis would remove theadhering bacteria.

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Gram staining depends on

Includes culture age, media, incubation

atmosphere, staining methods, . Similar

considerations apply to the interpretation of

smears from clinical specimens, and additionalfactors include different host cell types and

possible phagocytosis.

Gram stain permits the separation of all

bacteria into two large groups

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How the Gram Stain Work

So how does it work? Gram didn't know - hesimply worked empirically. We now know that theGram reaction is based on the structure of thebacterial cell wall.

In Gram-positive bacteria, the dark purple crystalviolet stain is retained by the thick layer ofpeptidoglycan which forms the outer layer of thecell.

In Gram-negative bacteria, the thin peptidoglycanlayer in the periplasm does not retain the darkstain, and the pink safranin counterstains thepeptidoglycan layer.

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Optimal use of Microscopy

Gram stained preparationshave to be observed withbright-field optics. Phase-contrast microscopydoes not allow therecognition of truecolours. Gram-positivebacteria may be seen underphase-contrast as red cells.Using bright-field optics,

Gram-positive cells arepurple or blue and Gram-negative pinkdue tocounter stain with Safranin..

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Report as follows

1 If no microorganisms are seen in asmear of a clinical specimen, reportNo microorganisms seen.

2. If microorganisms are seen, reportrelative numbers and Describemorphology.

Observe predominant shapes ofmicroorganisms

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A gram stained bacterial suspension containing a

mixture of Gram negative bacilli, and Gram positive

cocci arranged in bunches (Staphylococci spp)


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A true Gram Negative staining

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Value of Direct Smears

Guide the physician on initial choice ofantibiotic, pending results of culture andsensitivity.

Judge specimen quality.

Contribute to selection of culture media,especially with mixed flora.

Provide internal quality control whendirect smear results are compared toculture results.

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Staining depends on Structural

Integrity of Cell Wall

We know that only intact cells are Gram-

positive, so that cells which are even gently

broken become Gram-negative.

Observations suggest that bacterialprotoplasts, devoid of cell wall, are still

Gram-positive, indicating that it is probably

the semipermeable membrane which issomehow involved in the reaction.

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Nature of Morphology guides early Diagnosis

in uncommon diseases

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Identify

A young patient

presented with

foul smellingpurulent

discharge since 2

days onobservation by

Gram staining


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Gram stain of Neisseria gonorrhoeae,


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Observe Spores may appear as

Gram negative and Gram positive

Burkholderia pseudomallei is a gram negative


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Burkholderia pseudomallei is a gram-negative

bacilli with a safetypin appearance on

microscopic examination

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Limitations of Grams Staining

We know that Gram

positivity is

restricted almost

exclusively to thebacteria, with only a

few other groups,

such as the yeasts,exhibiting this

reaction.

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B tt U d t di f G


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Better Understanding of Grams

Staining

We should know that the Gram stain is

not an all-or-nothing phenomenon, but

thatquantitative variations

in Gram-

positivity exist between different

species,and within the same species

during different parts of the growthcycle or under different

environmental conditions.Dr.T.V.Rao MD 48


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Stains Several Fungi

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Streptococcus pneumonia

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Streptococcus pneumonia in Sputum

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Nocardia spp seen in Gram Staining

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Gram Stained Actinomyctes spp

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Faulty Gram stain reactions

It is possible to report as " Gram-

negative" if the gram-positive bacteria

are old, dead, or damaged and the

cell wall is not intact.

There is no equivalent "false Gram-

positive," but a false Gram-positivecan occur if the decolorization step is

accidentally omitted.Dr.T.V.Rao MD 54

Common errors in Staining


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Common errors in Staining

procedure Excessive heat during

fixation

Low concentration ofcrystal violet

Excessive washingbetween steps

Insufficient iodineexposure

Prolongeddecolourization

Excessive counterstaining

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G t i lt t l t d


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Gram stain results may not correlated

with culture results

Gram stain-positive, culture-negative

specimens may be the result of

contamination of reagents and other

supplies, presence of Antimicrobial

agents, or failure of organisms to grow

under usual Culture conditions (media,

atmosphere, etc.)

Presence of anaerobic microorganisms

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Artifacts in Gram Staining

Gram stainreagents CrystalViolet, Iodine ?,

Safranin,contaminated.

Dirty glass slides

Contaminatedwater used torinse slides

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Biochemical Tests in Identification

KOH string test may be

used as a confirmatory

test for the Gram Stain

(Powers, 1995, Arthi et

al., 2003): The

formation of a string

(DNA) in 3% KOH

indicates that theisolate is a gram-

negative organism.

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Gram staining not a fool proof


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Gram staining not a fool proof

procedure

Grams staining method

is not without its

problems.

It is , complicated, and

prone to operator

error.

The method also

requires a large number

of bacteria.Dr.T.V.Rao MD 59

Gram variable observations in Gram


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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

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Overcoming in Gram Variable


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Overcoming in Gram Variable

Observations

It is necessary that it is stainedat two or three different ages

(very young cultures should beused). In case a Gram-variablereaction is observed it is alsogood to check the purity of theculture.

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Gram Staining appearance differs..

The genera Actinomyctes,

Arthobacter,

Corynebacterium,

Mycobacterium, and

Propionibacterium have cellwalls particularly sensitive to

breakage during cell division,

resulting in Gram-negative

staining of these Gram-positive

cells. The staining of these

organisms result in an uneven

or granular appearance

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QUALITY CONTROL

Check appearance of reagents daily

If crystal violet has precipitate or crystalsediment, refilter before use even when

purchased commercially. NOTE: Some stains,especially basic fuchsin and safranin, canbecome contaminated. Start with freshmaterial in a clean bottle.

Evaporation may alter reagent effectiveness;working solutions should be changed regularly

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QUALITY CONTROL

Daily and when a new

lot is used, prepare a

smear ofEscherichia

coli(ATCC 25922) and

Staphylococcus

epidermidis (ATCC

12228)or

Staphylococcus aureus(ATCC 25923). Fix and

stain as described.

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Interpret Gram Staining with Clinical


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Interpret Gram Staining with Clinical

Picture and other Investigations

Nevertheless,

Gram's stain

findings can be

equivocal and,

therefore, must be

assessed carefully

in light of the

clinical picture.

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Modification in Gram staining


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Modification in Gram staining

methods ?

Since the original procedure ofGram, many variations of theGram staining technique havebeen published. Some of themhave improved the method,others include some minortechnical variants of no value.

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Modifications -Report with caution

Any final result is theoutcome of theinteraction of all ofthe possible

variables. All modified

methods to bepractised with

caution should suitto the laboratory,and quality controlchecks.

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Is it wise to adopt different Gram


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Is it wise to adopt different Gram

staining procedure

Bartholomew (1962) has

pointed out that each

variation in the Gram stainingprocedure has a definite limit

to its acceptability

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Hucker and Conn's


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Hucker and Conn s

recommendation

There is no gram procedure whichcan be referred to as the best for alllaboratories and for all situations. Itis recommended that the youngmicrobiologists adopt at least two ofthe well-accepted methods, practicethem until he is familiar with theircharacteristics,

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Words of Wisdom


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Words of Wisdom

Hans Christian Gram

I am aware

that as yet it

is very

defective

and

imperfectDr.T.V.Rao MD 70


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Creating Library of Gram Stains

Drain or gently blotexcess oil

For slide libraries andteaching collections that

will be stored for longerperiods, immersion oilcan be removed withxylene solution and the

slides can be coverslipped using Per mountto prevent fading.

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Best of References


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Best of ReferencesYou can read on line.

A monograph of

gram-stained

preparations of

clinical Specimens

By Linda M.

Marler, Jean A.Siders, Stephen D.

Allen (MD.)

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Gram staining continues to be


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Gram staining continues to be

Most Rapid test.

Even new molecular methodologiestypically take hours rather than minutes." This simple staining procedure

remains the most useful test performedin the microbiology lab. Results from aGram's stain can tell volumes about an

infection within 15 minutes of aspecimen's arrival in the lab, while mostother microbiology results require 24hours or more.

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Grams Staining


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Gram s Staining

A Mystery

The exact

mechanism of the

staining reaction is

not fully

understood,

however, this does

not detract from

its usefulness.

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gram staining guest lecture jipmer pondcherry

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