Gram Staining in

Clinical Microbiology
Dr.T.V.Rao. MD Professor of Microbiology Travancore Medical College, Kollam. Kerala

Dr.T.V.Rao MD

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

Tenth National Workshop on Simple Diagnostic Methods in Clinical Microbiology 29th November to 3rd December 2011

Department of Microbiology Jawaharlal Institute of Postgraduate Medical Education & Research, Pondicherry

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Working at Mansa General Hospital Mansa Republic of Zambia

Taught 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 later published this procedure in 1884. At the time, Dr. Gram was studying lung tissue sections from patients who had died of

pneumonia.
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First Paper on Gram Staining

In his paper, Dr. Gram described how he was able to visualize what we now call Staphylococcus, Streptococcus, Bacillus, and Clostridia in various histological sections. Interestingly, Dr. Gram did not actually use safranin as a counter stain in the original procedure (Gram negative cells would be colorless). He instead recommended using Bismarck brown as a counter stain to enable tissue cell nuclei to be visualized.
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Carl Weigert (1845-1904)

German pathologist Carl Weigert (18451904) from

Frankfurt, added a final step of staining with safranin.

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Traditional Definition of Gram stain

A method of staining bacteria using a violet stain. 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, washed with 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 use of the designation gram-positive should not be confused with the concept of staining cells with a simple stain that has a positive charge.
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Gram staining observation Basic Principle in Kochs postulations

The first of Kochs postulate that the suspected the organism should always be found in association with the disease.

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Poor quality of slides Can be corrected

Use of glass slides that have not been pre cleaned or degreased ?
NOTE: Storing slides in
a 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 a heat-fixed smear of a bacterial culture, followed by the addition of a mordant (Gram's iodine), rapid decolorization with alcohol 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 bacteria on a slide in a drop of water, allowing them to dry and then 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 Material

Wrong Right

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Using Methanol is it Better than Heat Fixation ?

Fix the smear with 95% Methanol Which will help in prevention of distortion of cells Helpful in Microscopic observation of CSF
and Urine
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Making Multiple smears in same slide

conserve resources
Making multiple smears make the optimal use of the slide. Reduces the economic costs and saves the technical time.
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Steps in Gram Staining ProcedureFollow the Clock

1 On

a rack, flood with filtered crystal violet ( Methyl violet ) 10 sec 2 Wash briefly in water to remove excess crystal violet 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 <6 seconds until the moving dye front has passed the lower edge of the section 6. Wash immediately in tap water 7. Counterstain with safranin for 15 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 violet stain will be removed from both G+ve & G-ve cells if the decolorizing agent(e.g alcohol ) is left on too long.
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Acetone used with Caution

Acetone is a more rapid decolorizes than alcohol and must be used with some care. Excessive decolorization turns Gram positive appear as Gram negative
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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, no known advantage can be gained by substituting the higher alcohols for ethyl alcohol.
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Step 6

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Which counterstain is better

Some bacteria which are poorly stained by Safranin, such as Hemophilus

spp., Legionella spp. , and some anaerobic

bacteria, are readily stained by basic fuchsin, but not Safranin
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Step 7

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Caring the stained slide

After the counterstain has been rinsed off, the slide is placed between some absorbent paper and the excess water gently blotted off. Care must be taken not to rub the slide with the blotting paper because this would remove the adhering 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 additional factors 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 - he simply worked empirically. We now know that the Gram reaction is based on the structure of the bacterial cell wall. In Gram-positive bacteria, the dark purple crystal violet stain is retained by the thick layer of peptidoglycan which forms the outer layer of the cell. In Gram-negative bacteria, the thin peptidoglycan layer in the periplasm does not retain the dark stain, and the pink safranin counterstains the peptidoglycan layer.
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Optimal use of Microscopy

Gram stained preparations have to be observed with bright-field optics. Phase-

contrast microscopy does not allow the recognition of true colours. Gram-positive

bacteria may be seen under phase-contrast as red cells. Using bright-field optics, Gram-positive cells are purple or blue and Gramnegative pink due to counter stain with Safranin..
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Report as follows
1 If no microorganisms are seen in a smear of a clinical specimen, report No microorganisms seen. 2. If microorganisms are seen, report relative numbers and Describe morphology.

Observe predominant shapes of microorganisms

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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)

A true Gram Negative staining

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Value of Direct Smears

Guide the physician on initial choice of antibiotic, pending results of culture and sensitivity. Judge specimen quality. Contribute to selection of culture media, especially with mixed flora. Provide internal quality control when direct smear results are compared to culture results.
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Staining depends on Structural Integrity of Cell Wall

We know that only intact cells are Grampositive, so that cells which are even gently broken become Gram-negative. Observations suggest that bacterial protoplasts, devoid of cell wall, are still Gram-positive, indicating that it is probably the semipermeable membrane which is somehow 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 smelling purulent discharge since 2 days on observation by Gram staining

Gram stain of Neisseria gonorrhoeae,

Observe Spores may appear as Gram negative and Gram positive

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 the bacteria, with only a few other groups, such as the yeasts, exhibiting this reaction.
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Better Understanding of Grams Staining

We should know that the Gram stain is not an all-or-nothing phenomenon, but that quantitative variations in Grampositivity exist between different species, and within the same species

during different parts of the growth cycle or under different environmental conditions.
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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 " Gramnegative" if the gram-positive bacteria are old, dead, or damaged and the cell wall is not intact. There is no equivalent "false Grampositive," but a false Gram-positive can occur if the decolorization step is accidentally omitted.
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Common errors in Staining procedure

Excessive heat during fixation Low concentration of crystal violet Excessive washing between steps Insufficient iodine exposure Prolonged decolourization
Excessive counterstaining
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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 stain reagents Crystal Violet, Iodine ?, Safranin, contaminated. Dirty glass slides Contaminated water used to rinse 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 the isolate is a gramnegative organism.
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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.
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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 Grampositive according to the conditions. With these types of organisms, Grampositive and Gram-negative cells may be present within the same preparation
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Overcoming in Gram Variable Observations

It is necessary that it is stained at two or three different ages (very young cultures should be used). In case a Gram-variable reaction is observed it is also good to check the purity of the culture.
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Gram Staining appearance differs..

The genera Actinomyctes, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls 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 crystal sediment, refilter before use even when purchased commercially. NOTE: Some stains, especially basic fuchsin and safranin, can become contaminated. Start with fresh material 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 of Escherichia 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 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 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.
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Modifications -Report with caution

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.
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Is it wise to adopt different Gram staining procedure

Bartholomew (1962) has pointed out that each variation in the Gram staining procedure has a definite limit to its acceptability
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Hucker and Conn's recommendation

There is no gram procedure which can be referred to as the best for all laboratories and for all situations. It is recommended that the young microbiologists adopt at least two of the well-accepted methods, practice them until he is familiar with their characteristics,
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Words of Wisdom Hans Christian Gram

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I am aware that as yet it is very defective and imperfect

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Creating Library of Gram Stains

Drain or gently blot excess oil
For slide libraries and teaching collections that will be stored for longer periods, immersion oil can be removed with xylene solution and the slides can be cover slipped using Per mount to prevent fading.
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Best of References
You 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 Most Rapid test.

Even new molecular methodologies typically take hours rather than minutes. " This simple staining procedure remains the most useful test performed in the microbiology lab. Results from a Gram's stain can tell volumes about an infection within 15 minutes of a specimen's arrival in the lab, while most other microbiology results require 24 hours or more.
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Grams 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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