Dr.T.V.Rao MD

Hans Christian Joachim Gram Danish Bacteriologist

Dyes become Stains

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

Gram staining a Important Technique

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 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 actylglucosamine monomers. In Gram positive bacteria, there are "wall teichoic acids". As well, between the cell wall and cell membrane, there is a "membrane teichoic

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.

The Cell walls differ…

Constituents of the Cell wall make difference

Definition of Gram stain

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 gramnegative.

Organizing the Staining Bottles

Method of Picking material from Agar plates Wrong Right

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

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 the slide kills the bacteria and makes sure that the bacteria a stuck to the slide and wont wash away during the staining procedure

Choosing a Right Smear

Before choosing a field for microscopic examination, it is important to look at the smear macroscopically Note that the smear is easily visible in ordinary

Requirements for Gram staining technique
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Glass slides (25 by 75 mm), frosted ends desirable b. 0.85% Nacl, sterile c. Pasteur pipettes and wood applicator sticks, sterile d. Microbiological loops, inoculating needles e. Supplies for disposal of biological waste, including “sharps” f. Bunsen burner g. Immersion oil

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.

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

Steps in Gram Staining Procedure- Follow the Clock

1 On

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a rack, flood with filtered crystal violet   10 sec 2 Wash briefly in water to remove excess crystal violet 3.   Flood with Gram’s iodine 10 sec 4.   Wash briefly in water, do not let the section dry out. 5.   Decolourise with acetone until the moving dye front has passed the lower edge of the section 6.   Wash immediately in tap water 7.   Note : If the section appears too blue repeat steps 6 and 7 8.   Counterstain with safranin 15 seconds

Proceed in organized Fashion

Step 1

Step 2

Step 3

Step 4

Step 5

Step 6

Step 7

Observe the Following Under Oil Immersion lens
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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

Microscopy: The Instruments

In a compound microscope the image from the objective lens is magnified again by the ocular lens. Total magnification = objective lens × ocular lens

Microscopy: The Instruments

Resolution is the ability of the lenses to distinguish two points. A microscope with a resolving power of 0.4 nm can distinguish between two points ≥ 0.4 nm. Shorter wavelengths of light provide

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. Grampositive bacteria may be seen under phasecontrast as red cells. Using bright-field optics, Gram-positive cells are purple or blue and Gram-

Use Bright field optics

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

Colors makes the Difference in Gram staining

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.

Observe with Caution to differentiate

Report as follows
 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

 Differential

Gram Stain Differentiates Gram positive from Gram negative
Stains: Gram Stain

Nature of Morphology in guides early Diagnosis in uncommon diseases

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.

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.

Mixed Flora of Staphylococcus and Streptococcus differentiates morphology

Stains Several Fungi

Gram-negative Pseudomonas aeruginosa bacteria (pinkrods).

(Bacillus anthracis),

Streptococcus pneumonia

Streptococcus salivarius

Gram stain of Neisseria gonorrhoea,

Streptococcus pneumoniae in Sputum

Clostridium spp as seen in Gram Stain

Moraxella as seen in Gram Staining

Clostridium difficile as seen by Gram staining

Corynebacterium diptheria as seen in Gram staining

Gram stain of Neisseria gonorrhoea,

Neisseria meningitides as seen in Gram staining

Nocardia spp seen in Gram Staining

Gram Stained Actinomyctes spp

Gram stained Listeria Monocytogenes

Gram Stained Cryptococcus neoformans

Artifacts in Gram Staining
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Gram stain reagents (Crystal Violet, Iodine, Safranin and Neutral Red Dirty glass slides Contaminated water used to rinse slides When investigating non-viable organisms seen in Gram stains it is always wise to eliminate every potential source.

Gram’s method stains Artificacts too

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 microorganism

Common errors in Staining procedure
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Excessive heat during fixation Low concentration of crystal violet Excessive washing between steps Insufficient iodine exposure Prolonged decolourization

Correlation of Gram stain with cultures
 Culture

results are not correlated with direct gram stain results.  Select true or false

1 True ( Correct )
2 False

Gram staining not a fool proof procedure

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

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 Gramnegative or Gram-positive according to the conditions. With these types of organisms, Gram-positive and Gramnegative 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 Gramvariable 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

Trouble shooting in Gram Staining method

It is important to note that gram-positivity (the ability to retain the purple crystal violet-iodine complex) is not an all-or-nothing phenomenon but a matter of degree. There are several factors which could result in a gram-positive organism staining gram-negatively 1. 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 violetiodine complex.

Trouble shooting in Gram Staining method

2. The age of the culture. Cultures more than 24 hours old may lose their ability to retain the crystal violet-iodine complex. 3. The organism itself. Some grampositive bacteria are more able to retain the crystal violet-iodine complex than others.  One must use very precise techniques in gram staining and interpret the results with discretion

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.

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.

Gram staining continues to be Most Rapid test.

Even new molecular methodologies typically take hours rather than minutes. Fortunately, Gram's stain, devised by a Danish pathologist in 1884, exists (see "The man behind Gram's stain, and "A revolution in staining begins," 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. Nevertheless, Gram's stain findings can be equivocal and, therefore, must be assessed carefully in light of the clinical picture.

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Dr.T.V.Rao MD Email doctortvrao@gmail.com

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