gram staining in diagnostic microbiology

GGRRAAMM STAININGSTAINING

Dr.T.V.Rao MDDr.T.V.Rao MD

Dr.T.V.Rao MDDr.T.V.Rao MD 11

Hans Christian Joachim Hans Christian Joachim GramGram

Danish BacteriologistDanish Bacteriologist


Dyes become StainsDyes become Stains

With interest in the effects of dyes on With interest in the effects of dyes on living tissue. In 1884 the living tissue. In 1884 the DanishDanish microbiologistmicrobiologist Hans Christian Gram Hans Christian Gram discovered that crystal violet discovered that crystal violet irreversibly stains certain bacteria but irreversibly stains certain bacteria but can be washed from others. The dye can be washed from others. The dye has been widely used ever since for the has been widely used ever since for the Gram stain technique, which identifies Gram stain technique, which identifies bacteria as gram-positive (the stain is bacteria as gram-positive (the stain is retained) or gram-negative (the stain is retained) or gram-negative (the stain is washed). washed).


Gram staining a Important Gram staining a Important TechniqueTechnique

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


Gram positive BacteriaGram positive Bacteria Gram positive bacteria Gram positive bacteria

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


Gram Negative BacteriaGram Negative Bacteria Gram negative bacteria Gram negative bacteria

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


The Cell walls differ…The Cell walls differ…


Constituents of the Cell Constituents of the Cell wall make differencewall make difference


Definition of Gram stainDefinition of Gram stain

A method of staining bacteria using a A method of staining bacteria using a violet stain. The gram staining violet stain. The gram staining characteristics (denoted as positive or characteristics (denoted as positive or negative). A heat fixed bacterial smear is negative). A heat fixed bacterial smear is stained with crystal violet (methyl stained with crystal violet (methyl violet), treated with 3% iodine/potassium violet), treated with 3% iodine/potassium iodide solution, washed with alcohol and iodide solution, washed with alcohol and counterstained. The method counterstained. The method differentiates bacteria into two main differentiates bacteria into two main classes, gram-positive and gram-classes, gram-positive and gram-negative.negative.


Organizing the Staining Organizing the Staining BottlesBottles


Method of Picking material Method of Picking material from Agar platesfrom Agar plates

Wrong Wrong RightRight


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


Making a SmearMaking a Smear First prepare your slide. First prepare your slide.

You do this by placing You do this by placing bacteria on a slide in a bacteria on a slide in a drop of water, allowing drop of water, allowing them to dry and then them to dry and then heat fixing them. heat fixing them. Heating the slide kills Heating the slide kills the bacteria and makes the bacteria and makes sure that the bacteria a sure that the bacteria a stuck to the slide and stuck to the slide and wont wash away during wont wash away during the staining procedure the staining procedure


Choosing a Right Choosing a Right SmearSmear

Before choosing a Before choosing a field for microscopic field for microscopic examination, it is examination, it is important to look at important to look at the smear the smear macroscopically macroscopically

Note that the smear Note that the smear is easily visible in is easily visible in ordinary light ordinary light


Requirements for Gram Requirements for Gram staining techniquestaining technique

Glass slides (25 by 75 mm), frosted ends Glass slides (25 by 75 mm), frosted ends desirabledesirable

b. 0.85% Nacl, sterileb. 0.85% Nacl, sterile c. Pasteur pipettes and wood applicator c. Pasteur pipettes and wood applicator

sticks, sterilesticks, sterile d. Microbiological loops, inoculating needlesd. Microbiological loops, inoculating needles e. Supplies for disposal of biological waste, e. Supplies for disposal of biological waste,

including “sharps”including “sharps” f. Bunsen burnerf. Bunsen burner g. Immersion oilg. Immersion oil


Making Multiple smears in Making Multiple smears in same slide – conserve same slide – conserve

resourcesresources Making multiple Making multiple

smears make the smears make the optimal use of the optimal use of the slide.slide.

Reduces the Reduces the economic costs economic costs and saves the and saves the technical time.technical time.


Correct preparationCorrect preparation

Smear preparation: Proper smear Smear preparation: Proper smear preparation should produce a preparation should produce a monolayer of organisms sufficiently monolayer of organisms sufficiently dense for easy visualization but thin dense for easy visualization but thin enough to reveal characteristic enough to reveal characteristic morphological characteristics. Use morphological characteristics. Use clean, new glass slides. clean, new glass slides.

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

culture media firstculture media first


Steps in Gram Staining Steps in Gram Staining Procedure- Procedure- Follow the ClockFollow the Clock 11 On a rack, flood with filtered crystal On a rack, flood with filtered crystal

violet 10 secviolet 10 sec 2 Wash briefly in water to remove excess 2 Wash briefly in water to remove excess

crystal violetcrystal violet 3. Flood with Gram’s iodine 10 sec3. Flood with Gram’s iodine 10 sec 4. Wash briefly in water, do not let the 4. Wash briefly in water, do not let the

section dry out. section dry out. 5. Decolourise with acetone until the 5. Decolourise with acetone until the

moving dye front has passed the lower edge moving dye front has passed the lower edge of the sectionof the section

6. Wash immediately in tap water6. Wash immediately in tap water 7. Note : If the section appears too blue 7. Note : If the section appears too blue

repeat steps 6 and 7repeat steps 6 and 7 8. Counterstain with safranin 15 seconds8. Counterstain with safranin 15 seconds


Proceed in organized Proceed in organized FashionFashion


Step 1Step 1


Step 2Step 2


Step 3Step 3


Step 4Step 4


Step 5Step 5


Step 6Step 6


Step 7Step 7


Observe the Following Observe the Following Under Oil Immersion lensUnder Oil Immersion lens

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

ii. Relative amounts of squamous epithelial ii. Relative amounts of squamous epithelial cells, bacteriacells, bacteria

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

iii. Location and arrangements of iii. Location and arrangements of microorganismsmicroorganisms

Note any special character sticks Note any special character sticks


Microscopy: The Microscopy: The InstrumentsInstruments

In a compound In a compound microscope the microscope the image from the image from the objective lens is objective lens is magnified again by magnified again by the ocular lens.the ocular lens.

Total magnification Total magnification ==objective lens objective lens ocular lensocular lens


Microscopy: The Microscopy: The InstrumentsInstruments

Resolution is the Resolution is the ability of the lenses ability of the lenses to distinguish two to distinguish two points.points.

A microscope with a A microscope with a resolving power of resolving power of 0.4 nm can 0.4 nm can distinguish between distinguish between two points ≥ 0.4 nm.two points ≥ 0.4 nm.

Shorter wavelengths Shorter wavelengths of light provide of light provide greater resolution.greater resolution.


Optimal use of Optimal use of MicroscopyMicroscopy

Gram stained preparations Gram stained preparations have to be observed with have to be observed with bright-field optics. bright-field optics. Phase-Phase-contrast microscopy contrast microscopy does not allow the does not allow the recognition of true recognition of true colourscolours. Gram-positive . Gram-positive bacteria may be seen bacteria may be seen under phase-contrast as under phase-contrast as red cells. Using bright-field red cells. Using bright-field optics, Gram-positive cells optics, Gram-positive cells are purple or blue and are purple or blue and Gram-negative Gram-negative pink pink due to due to counter stain with counter stain with safranin.. safranin..


Use Bright field opticsUse Bright field optics

With bright-field With bright-field optics colours optics colours can be can be discriminated discriminated best if the best if the condenser iris is condenser iris is opened as far as opened as far as possible without possible without discomfort to the discomfort to the eyeseyes


Colors makes the Colors makes the Difference in Gram stainingDifference in Gram staining

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

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


Observe with Caution to Observe with Caution to differentiatedifferentiate


Report as followsReport as follows

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

specimen, report “No specimen, report “No microorganisms seen.”microorganisms seen.”

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

Describe morphology.Describe morphology. Observe predominant shapes of Observe predominant shapes of

microorganismsmicroorganisms


Gram Stain Differentiates Gram Stain Differentiates Gram positive from Gram Gram positive from Gram

negativenegative Differential Stains: Gram StainDifferential Stains: Gram Stain


Nature of Morphology in Nature of Morphology in guides early Diagnosis in guides early Diagnosis in

uncommon diseasesuncommon diseases


Creating Library of Gram Creating Library of Gram StainsStains

Drain or gently blot Drain or gently blot excess oilexcess oil

For slide libraries and For slide libraries and teaching collections teaching collections that will be stored for that will be stored for longer periods, longer periods, immersion oil can be immersion oil can be removed with xylene removed with xylene solution and the slides solution and the slides can be cover slipped can be cover slipped using Per mount to using Per mount to prevent fading.prevent fading.


Value of Direct SmearsValue of Direct Smears

Guide the physician on initial choice Guide the physician on initial choice of antibiotic, pending results of of antibiotic, pending results of culture and sensitivity.culture and sensitivity.

Judge specimen quality.Judge specimen quality. Contribute to selection of culture Contribute to selection of culture

media, especially with mixed flora.media, especially with mixed flora. Provide internal quality control when Provide internal quality control when

direct smear results are compared to direct smear results are compared to culture results.culture results.


Mixed Flora of Staphylococcus Mixed Flora of Staphylococcus and Streptococcusand Streptococcus

differentiates morphologydifferentiates morphology


Stains Several FungiStains Several Fungi


Gram-negative Gram-negative Pseudomonas Pseudomonas aeruginosaaeruginosa bacteria bacteria (pink-(pink-

rods).rods).


((Bacillus anthracisBacillus anthracis),),


Streptococcus Streptococcus pneumoniapneumonia


Streptococcus salivariusStreptococcus salivarius


Gram stainGram stain of Neisseria of Neisseria gonorrhoea,gonorrhoea,


Streptococcus pneumoniaeStreptococcus pneumoniae in Sputumin Sputum


Clostridium spp as seen in Gram Clostridium spp as seen in Gram Stain Stain


Moraxella as seen in Gram Moraxella as seen in Gram StainingStaining


Clostridium difficile as seen by Clostridium difficile as seen by Gram stainingGram staining


Corynebacterium diptheria as Corynebacterium diptheria as seen in Gram stainingseen in Gram staining


Gram stainGram stain of Neisseria of Neisseria gonorrhoea,gonorrhoea,


Neisseria meningitides as seen Neisseria meningitides as seen in Gram stainingin Gram staining


Nocardia spp seen in Gram Nocardia spp seen in Gram StainingStaining


Gram Stained Actinomyctes Gram Stained Actinomyctes sppspp


Gram stained Listeria Gram stained Listeria MonocytogenesMonocytogenes


Gram Stained Gram Stained Cryptococcus Cryptococcus neoformansneoformans


Artifacts in Gram Artifacts in Gram StainingStaining

Gram stain reagents (Crystal Violet, Gram stain reagents (Crystal Violet, Iodine, Safranin and Neutral RedIodine, Safranin and Neutral Red

Dirty glass slidesDirty glass slides Contaminated water used to rinse slidesContaminated water used to rinse slides When investigating non-viable organisms When investigating non-viable organisms

seen in Gram stains it is always wise to seen in Gram stains it is always wise to eliminate every potential source. eliminate every potential source.


Gram’s method stains Gram’s method stains Artificacts tooArtificacts too


Gram stain results may not Gram stain results may not correlated with culture resultscorrelated with culture results

Gram stain-positive, culture-negative Gram stain-positive, culture-negative specimens may be the result of specimens may be the result of contamination of reagents and other contamination of reagents and other supplies, presence ofsupplies, presence of

Antimicrobial agents, or failure of Antimicrobial agents, or failure of organisms to grow under usual Culture organisms to grow under usual Culture conditions (media, atmosphere, etc.)conditions (media, atmosphere, etc.)

Presence of anaerobic Presence of anaerobic microorganismmicroorganism


Common errors in Staining Common errors in Staining procedureprocedure

Excessive heat during Excessive heat during fixation fixation

Low concentration of Low concentration of crystal violetcrystal violet

Excessive washing Excessive washing between steps between steps

Insufficient iodine Insufficient iodine exposure exposure

Prolonged Prolonged decolourization decolourization

Excessive counterstainingExcessive counterstaining


Correlation of Gram stain Correlation of Gram stain with cultureswith cultures

Culture results are not correlated Culture results are not correlated with direct gram stain results.with direct gram stain results.

Select true or falseSelect true or false

1 True 1 True ( Correct( Correct ) )

2 False2 False


Gram staining not a fool Gram staining not a fool proof procedureproof procedure

Gram’s staining Gram’s staining method is not without method is not without its problems. It is , its problems. It is , complicated, and complicated, and prone to operator prone to operator error.error. The method The method also requires a large also requires a large number of cells number of cells However, it is also However, it is also central to phenotypic central to phenotypic microbial identification microbial identification techniques. techniques.


Gram variable observations Gram variable observations in Gram stainingin Gram staining

The Gram staining procedure does The Gram staining procedure does not always give clear-cut results. not always give clear-cut results. Some organisms are Gram-variable Some organisms are Gram-variable and may appear either Gram-and may appear either Gram-negative or Gram-positive according negative or Gram-positive according to the conditions. With these types of to the conditions. With these types of organisms, Gram-positive and Gram-organisms, Gram-positive and Gram-negative cells may be present within negative cells may be present within the same preparation the same preparation


Overcoming in Gram Overcoming in Gram Variable ObservationsVariable Observations

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


Why Gram VariabilityWhy Gram Variability??

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


Trouble shooting in Gram Trouble shooting in Gram Staining methodStaining method

It is important to note that gram-positivity (the It is important to note that gram-positivity (the ability to retain the purple crystal violet-iodine ability to retain the purple crystal violet-iodine complex) is not an all-or-nothing phenomenon but complex) is not an all-or-nothing phenomenon but a matter of degree. There are a matter of degree. There are several factors several factors which could result in a gram-positive which could result in a gram-positive organism staining gram-negativelyorganism staining gram-negatively

1. 1. The method and techniques used.The method and techniques used.

Overheating during heat fixation, over Overheating during heat fixation, over decolourization with alcohol, and even too much decolourization with alcohol, and even too much washing with water between steps may result in washing with water between steps may result in gram-positive bacteria losing the crystal violet-gram-positive bacteria losing the crystal violet-iodine complex. iodine complex.


Trouble shooting in Gram Trouble shooting in Gram Staining methodStaining method

2. 2. The age of the cultureThe age of the culture. . Cultures more Cultures more than 24 hours old may lose their ability to than 24 hours old may lose their ability to retain the crystal violet-iodine complex. retain the crystal violet-iodine complex.

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

One must use very precise One must use very precise techniques in gram staining and techniques in gram staining and

interpret the results with discretioninterpret the results with discretion


Poor quality of slidesPoor quality of slidesCan be correctedCan be corrected

Use of glass slides Use of glass slides that have not been that have not been pre cleaned or pre cleaned or degreased degreased NOTENOTE: : Storing slides in a Storing slides in a jar with 95% jar with 95% ethanol will ensure ethanol will ensure clean slides. Drain clean slides. Drain excess alcohol or excess alcohol or flame slide before flame slide before use.use.


Modification in Gram Modification in Gram staining methods ?staining methods ?

Since the original procedure of Gram, many Since the original procedure of Gram, many variations of the Gram staining technique variations of the Gram staining technique have been published. Some of them have have been published. Some of them have improved the method, others include some improved the method, others include some minor technical variants of no value. minor technical variants of no value. Bartholomew (1962) has pointed out that Bartholomew (1962) has pointed out that each variation in the Gram staining procedure each variation in the Gram staining procedure has a definite limit to its acceptability. Any has a definite limit to its acceptability. Any final result is the outcome of the interaction final result is the outcome of the interaction of all of the possible variables.of all of the possible variables.

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


Gram staining continues to Gram staining continues to be Most Rapid testbe Most Rapid test..

Even new molecular methodologies typically take Even new molecular methodologies typically take hours rather than minutes. Fortunately, Gram's hours rather than minutes. Fortunately, Gram's stain, devised by a Danish stain, devised by a Danish pathologistpathologist in 1884, in 1884, exists (see "The man behind Gram's stain, and "A exists (see "The man behind Gram's stain, and "A revolution in staining begins," This simple revolution in staining begins," This simple staining procedure remains the most useful test staining procedure remains the most useful test performed in the microbiology lab. Results from a performed in the microbiology lab. Results from a Gram's stain can tell volumes about an infection Gram's stain can tell volumes about an infection within 15 minutes of a specimen's arrival in the within 15 minutes of a specimen's arrival in the lab, while most other microbiology results require lab, while most other microbiology results require 24 hours or more. Nevertheless, Gram's stain 24 hours or more. Nevertheless, Gram's stain findings can be equivocal and, therefore, must be findings can be equivocal and, therefore, must be assessed carefully in light of the clinical picture. assessed carefully in light of the clinical picture.


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