Gram Staining Skill Based Learning

Gram’s Stainingskill based learning Dr.T.V.Rao. MD Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

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 Clostridiain 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. Dr.T.V.Rao MD

Carl Weigert (1845-1904) • Gram did not use a counterstain in his procedure. It was a few years later, that the German pathologist Carl Weigert (1845-1904) from Frankfurt, added a final step of staining with safranin. Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

The Cell walls differ… Dr.T.V.Rao MD

Gram Positive Structure Peptidoglycan Cytoplasmic Membrane SectionEnlargement = Cytoplasm Cell Cell Wall Within the peptidoglycan stand are teichoic acids and polysaccharides

Gram Negative Structure Outer Membrane Peptidoglycan SectionEnlargement Cytoplasmic Membrane = Cytoplasm Cell Cell Wall The outer membrane contains Porin trimers, O antigens and Lipopolysaccharides. There is also a space between the Cytoplasmic Membrane and the Outer Membrane which is known as the Periplasmic Space

Gram staining observation Basic Principle in Koch’s postulations • The first of Koch’s postulatethat the suspected the organism should always be found in association with the disease. Dr.T.V.Rao MD

Requirements for Gram staining technique • 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 Dr.T.V.Rao MD

Poor quality of slidesCan 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. Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

Organizing the Staining Bottles Dr.T.V.Rao MD

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 Dr.T.V.Rao MD

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 Dr.T.V.Rao MD

Method of Picking material from Agar platesWrong Right Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

Steps in Gram Staining Procedure- Follow the Clock • 1On a rack, flood with filtered crystal violet ( Methyl 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. Counterstain with safranin 15 seconds Dr.T.V.Rao MD

Proceed in organized Fashion Dr.T.V.Rao MD

Step 1 Dr.T.V.Rao MD

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 Dr.T.V.Rao MD

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. Kisskalt (84) found decolorization power decreasing in the following order: methyl, ethyl, propyl, butyl, and amyl alcohol. Conn found a mixture of equal parts of methyl and isopropyl alcohols to have very similar decolorization properties to ethyl alcohol. In practice, however, no known advantage can be gained by substituting the higher alcohols for ethyl alcohol. Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

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 Gram-negative pinkdue to counter stain with Safranin.. Dr.T.V.Rao MD

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 Dr.T.V.Rao MD

Gram Staining – Gram +’ve Dr.T.V.Rao MD

Gram Staining – Gram -’ve Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

Nature of Morphology in guides early Diagnosis in uncommon diseases Dr.T.V.Rao MD

Limitations of Gram’s Staining • We know that Gram positivityis restricted almost exclusively to the bacteria, with only a few other groups, such as the yeasts, exhibiting this reaction. Dr.T.V.Rao MD

Better Understanding of Gram’s Staining • We know that the Gram stain is not an all-or-nothing phenomenon, but that quantitative variations in Gram-positivity exist between different species, and within the same species during different parts of the growth cycle or under different environmental conditions. We know that only intact cells are Gram-positive, so that cells which are even gently broken become Gram-negative. We know 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. Dr.T.V.Rao MD

Stains Several Fungi Dr.T.V.Rao MD

Streptococcus pneumonia Dr.T.V.Rao MD

Streptococcus pneumonia in Sputum Dr.T.V.Rao MD

Gram stain of Neisseria gonorrhoea, Dr.T.V.Rao MD

Nocardia spp seen in Gram Staining Dr.T.V.Rao MD

Gram Stained Actinomyctes spp Dr.T.V.Rao MD

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 Dr.T.V.Rao MD

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 Dr.T.V.Rao MD

Artifacts in Gram Staining • 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. Dr.T.V.Rao MD

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. Dr.T.V.Rao MD

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 Dr.T.V.Rao MD

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 just visible. Dr.T.V.Rao MD

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 Gram-variable reaction is observed it is also good to check the purity of the culture. Dr.T.V.Rao MD

Gram Staining Skill Based Learning