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Gram staining continues to be important procedure to diagnose several bacterial infection

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G r a m staining


Dr.T.V.Rao MD

Hans christian joachim gram danish bacteriologist
Hans Christian Joachim GramDanish Bacteriologist

Dyes become stains
Dyes become Stains

  • With interest in the effects of dyes on living tissue. In 1884 the DanishmicrobiologistHans 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 gram-negative (the stain is washed).

Gram staining a important technique
Gram staining a Important Technique

  • A 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

  • 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 acid".

Gram negative bacteria
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.

Definition of gram stain
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.

Making a smear
Making a Smear glass slide

  • 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
Choosing a Right Smear glass slide

  • 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 light

Requirements for gram staining technique
Requirements for Gram staining technique glass slide

  • 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 in same slide – conserve resources glass slide

  • Making multiple smears make the optimal use of the slide.

  • Reduces the economic costs and saves the technical time.

Correct preparation
Correct preparation glass slide

  • 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
Steps in Gram Staining Procedure- glass slideFollow the Clock

  • 1On 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

Step 1
Step 1 glass slide

Step 2
Step 2 glass slide

Step 3
Step 3 glass slide

Step 4
Step 4 glass slide

Step 5
Step 5 glass slide

Step 6
Step 6 glass slide

Step 7
Step 7 glass slide

Observe the following under oil immersion lens
Observe the Following Under Oil Immersion lens glass slide

  • 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
Microscopy: The Instruments glass slide

  • 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 instruments1
Microscopy: The Instruments glass slide

  • 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 greater resolution.

Optimal use of microscopy
Optimal use of Microscopy glass slide

  • 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 Gram-negative pink due to counter stain with safranin..

Use bright field optics
Use Bright field optics glass slide

  • 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
Colors makes the Difference in Gram staining glass slide

  • 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 secondreddye have got both a cell wall and a cell membrane - they are called Gram negative.

Report as follows
Report as follows glass slide

  • 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

Gram stain differentiates gram positive from gram negative
Gram Stain Differentiates Gram positive from Gram negative glass slide

  • Differential Stains: Gram Stain

Creating library of gram stains
Creating Library of Gram Stains diseases

  • 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
Value of Direct Smears diseases

  • 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
Mixed Flora of Staphylococcus and Streptococcus diseasesdifferentiates morphology

Gram negative pseudomonas aeruginosa bacteria pink rods
Gram-negative diseasesPseudomonas aeruginosa bacteria (pink-rods).

Bacillus anthracis
( diseasesBacillus anthracis),

Gram stain of neisseria gonorrhoea
Gram stain diseases of Neisseria gonorrhoea,

Streptococcus pneumoniae in sputum
Streptococcus pneumoniae diseases in Sputum

Gram stain of neisseria gonorrhoea1
Gram stain diseases of Neisseria gonorrhoea,

Gram stained cryptococcus neoformans
Gram Stained diseasesCryptococcus neoformans

Artifacts in gram staining
Artifacts in Gram Staining diseases

  • 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 stain results may not correlated with culture results
Gram stain results may not correlated with culture results diseases

  • 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
Common errors in Staining procedure diseases

  • Excessive heat during fixation

  • Low concentration of crystal violet

  • Excessive washing between steps

  • Insufficient iodine exposure

  • Prolonged decolourization

  • Excessive counterstaining

Correlation of gram stain with cultures
Correlation of Gram stain with cultures diseases

  • 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 staining not a fool proof procedure diseases

  • 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
Gram variable observations in Gram staining diseases

  • 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

Overcoming in gram variable observations
Overcoming in Gram Variable Observations diseases

  • 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 Gram-variable reaction is observed it is also good to check the purity of the culture.

Why gram variability
Why Gram Variability diseases?

  • 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 just visible.

Trouble shooting in gram staining method
Trouble shooting in Gram Staining method diseases

  • 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 violet-iodine complex.

Trouble shooting in gram staining method1
Trouble shooting in Gram Staining method diseases

  • 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 gram-positive 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
Poor quality of slides diseasesCan 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
Modification in Gram staining methods ? diseases

  • 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
Gram staining continues to be Most Rapid test diseases.

  • 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.

Created for e learning to medical and paramedical students in developing countreis

Created for ‘e’ learning to Medical and Paramedical students in developing countreis

Dr.T.V.Rao MD


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