smear prep & Gram staining-new.pptx
- 1. SMEAR PREPARATION & GRAM STAINING Dr.priyadharshini.S , 1st year postgraduate , Department of microbiology , Chettinad hospital and research institute ,
- 2. SMEAR PREPARATION SMEAR should be spread evenly covering an area of about 15 - 20mm diameter on a slide. The technique used to make smears from different specimens are as follows: ■ Purulent specimen: using a sterile wire loop, make a thin preparation.donot centrifuge a purulent fluid eg c s.f containing pus cells. ■ Non purulent fluid specimen: centrifuge the fluid and make a smear from a drop of the well mixed sediment. ■ Culture: Emulsify a colony in sterile distilled water and make a thin preparation on a slide, when a broth culture transfer a loop ful to a slide and make a thin preparation. ■ Sputum : use a piece of clean stick to transfer and spread purulent and caseous material on a slide. Soak the stick in a phenol Or hypochlorite disinfectant before discarding it. ■ Swabs: Roll the swab Ona slide. This is particularly important when looking for intracellular bacteria such as N.gonorrhoea ( urtheral, cervical, eye swab). Rolling the swab avoids damaging the pus cells. ■ Faeces: Use apiece of clean stick to transfer pus and mucus to a slide decontaminate the stick before discarding it.spread to make a thin preparation.
- 3. DRYING AND FIXING SMEARS ■ After making a smear,leave the slide in a safe place For the smear to air dry, ■ When a smear requires urgent staining, it can be dried quickly using gentle heat ■ The purpose of fixation is to preserve microorganisms and to prevent smear being washed from slides during staining. ■ Smears are fixed by heat, alcohol, or occasionally by other chemicals.
- 4. HEAT FIXATION: ■ This is widely used but can damage organisms and alter their staining reactions especially when excessive heat is used. Heat fixation also damages leucocytes and is therefore unsuitable for fixing smears which may contain intracellular organisms such as N. Gonorrhoeae and N. Meningitidis. ■ When used, heat fixation must be carried out with care. The following technique is recommended: ■ Allow the smear to air-dry completely. ■ Rapidly pass the slide, smear uppermost, three times through the flame of a spirit lamp or pilot flame of a Bunsen burner. ■ Note: After passing the slide through the flame three times, it should be possible to lay the slide on the back of the hand without the hand feeling uncomfortably hot. When this cannot be done, too much heat has been used.
- 5. ALCOHOL FIXATION ■ This form of fixation is far less damaging to microorganisms than heat. ■ Cells especially pus cells, are also well preserved. ■ Alcohol fixation is therefore recommended for fixing smears when looking for gram negative intracellular diplococci. ■ Alcohol fixation is more bactericidal than heat ( eg: M.tuberculosis is rapidly killed in sputum smears after applying 70% alcohol) ■ A method of alcohol fixation is as follows: 1. Allow the smear to air dry completely. 2. Depending on the Type of smear, alcohol fix as follows: – For the detection of intracellular gram negative diplococci (N.gonorrhoeae or
- 6. CHEMICAL FIXATIVES ■ Other chemicals are some times necessary to fix smears which contains particularly dangerous organisms to ensure all the organisms are killed eg 40g/l potassium permaganate is recommended for fixing smears which may contain Anthrax bacilli. ■ Formaldehyde vapour is sometimes recommended for fixing smears which may contain MYCOBACTERIUM species. ■ Formaldehyde fixed smears however tend to stain poorly and the chemical itself is toxic with an injurious vapour.
- 7. GRAM STAINING •Hans Christian Joachim Gram ( 13 September 1853 – 14 November 1938)
- 8. CORNER STONE OF BACTERIAL INDENTIFICATION ■ Danish physician Hans Christian Gram developed the Gram staining method in 1884. ■ Gram staining is still the cornerstone of bacterial identification and taxonomic division. Besides demonstrating the morphology of bacteria ,the gram stain can be used to divide most bacterial species into 2 groups. ■ Those that take up the basic dye ( crystal violet) is GRAM POSITIVE ■ Those that allow the crystal violet dye to washout easily with decolourizer (Alcohol or Acetone) is GRAM NEGATIVE ■ Gram-positive bacteria- stains Darkpurple ■ Gram-negative bacteria-stains red/pink
- 9. PRINCIPLES OF GRAM STRAINING ■ CELL WALL THEORY:The differences in Gram-positive and Gram-negative bacteria cell wall composition account for the Gram staining differences. Gram-positive cell wall contains a thick layer of peptidoglycan with numerous teichoic acid cross-linking, which resists decolorization. ■ PH THEORY: In aqueous solutions, crystal violet dissociates into CV+ and Cl – ions that penetrate through Gram-positive and Gram-negative cell walls. The CV+ interacts with negatively charged componeIn contrast, the nts of bacterial cells, staining the cells purple. When added, iodine (I- or I3-) interacts with CV+ to form large crystal violet-iodine (CV-I) complexes within the cytoplasm and outer layers of the cell. ■ The decolorizing agent (ethanol or an ethanol and acetone solution) interacts with the lipids of both gram-positive and gram-negative bacteria membranes. ■ The outer membrane of the Gram-negative cell is lost from the cell, leaving the thin peptidoglycan layer exposed. With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell. ■ The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell is dehydrated by the addition of ethanol. Thus ethanol treatment traps the large CV-I complexes within the cell. ■ After decolorization, the gram-positive cell remains purple. In contrast, the gram-
- 10. Cell wall of Gram-positive and Gram-negative Bacteria
- 12. All cocci are gram positive EXCEPT: 1. Meningococci 2. Gonococci 3. Veilonella 4. Moraxella All bacilli are gram negative Except: 1. MYCOBACTERIUM 2. ANTHRACIS BACILLUS 3. CLOSTRIDIUM SP 4. DIPTHERIAE CORYNEBACTERIUM 5. NOCARDIA 6. ACTINOMYCETES 7. LISTERIA 8. DIPTHEROIDS
- 13. STEPS OF GRAM STAINING ■ Fixation of clinical materials made on a slide from bacterial culture or specimen is air dried and then heat fixed. ■ STEP 1:Application of the primary stain (crystal violet) for 1 minute. Then he slide is rinsed with water.Crystal violet is a dark blue to purple dye. It stains all cells blue/purple. ■ STEP 2:Application of mordant: The Gram’s iodine solution (mordant) is Poured over the slide for 1 minute.then the slide is rinsed with water.Grams iodine form a crystal violet-iodine (CV-I) complex; all cells continue to appear blue. ■ STEP 3:Decolorization : Pouring few drops of decolourizer to the smear eg: acetone for 1-2sec or ethyl alcohol 20-30sec or Acetone alcohol for 10sec.slide is immediately rinsed with water.The decolorization step distinguishes gram-positive from gram-negative cells. The organic solvent such as acetone or ethanol extracts the blue dye complex from the lipid-rich, thin-walled gram-negative bacteria to a greater degree than from the lipid-poor, thick-walled, gram-
- 14. Note: Decolorization is the most crucial step of gram staining.if the decolourizer is Poured for more times even gram positive bacteria lose color (Over Decolorization) And if poured less timethe gram negative bacteria donot lose the primary color of primary stain properly ( under Decolorization) ■ STEP 4: Application of counterstain (safranin) is added for 1 minute.then the slide is rinse with water. The red dye safranin stains the decolorized gram-negative cells red/pink; the gram-positive bacteria remain blue. The slide is dried and then examined under oil immersion objective.
- 15. • After performing a gram stain, the technician should first determine whether the Gram stain is adequate. In an appropriately stained biological specimen, the nuclei of neutrophils are red. If the nuclei are blue, the decolorization is insufficient. Results ■ Gram-negative bacteria will stain pink/red and ■ Gram-positive bacteria will stain blue/purple.
- 16. REAGENTS: violet dye: ■ Crystal violet or methyl violet is used as a concentration of 0.5 to 2 %. 1. Crystal violet 10g 2. Absolute alcohol 100ml 3. Distilled water 1litre ■ Dissolve the dye in the alcohol, filter through filter paper and add to the water.
- 17. ■ However, gram positive staining can be strengthened by the addition of sodium bicarbonate or ammonium oxalate as in the following solutions: 1. Kopeloff & Beerman’s stain : solution A: methylviolet 10g Distilled water 1litre Solution B: sodium bicarbonate 50g . Distilled water 1 litre Shortly before use mix 30 volumes of solution A with 8 volume of solution B It is a disadvantage of this mixture that it tends to precipitate within a few days and so cannot be kept. 2. Preston & Morrells stain: . Crystal violet 20g, methylated spirit 200ml, Ammonium oxalate 1% in water 800 ml
- 18. IODINE SOLUTION ■ To obtain iodine in aqueous solution, potassium iodide or sodium hydroxide must be added. ■ The more alkaline solution with sodium hydroxide is thought to give slightly stronger gram positive staining. ■ Gram’s iodine (lugol’s): . Iodine 10g Potassium iodide 20g Distilled water 1 litre Kopeloff & Beerman’s iodine: Iodine 20 g Sodium hydroxide (4%NAOH) 100ml Distilled water 900ml
- 19. DECOLOURIZER: ■ ACETONE: 1. this is the fastest and most specific decolourizer. 2. It is applied to the smear for only 2-3 Seconds and to sections for a second or 2 longer. 3. It’s rapidity is an advantage when only a single slide is to be stained. 4. However the short period of exposure is difficult to control when many slides have to be stained simultaneously.
- 20. ABSOLUTE ALCOHOL(100%ETHANOL) ■ This acts more slowly than acetone and should be applied and reapplied for about 1 min until on tilting the slide from slide to slide . ACETONE ALCOHOL: ■ This is a mixture of 1 volume of acetone with 1 volume of 95% ethanol. ■ It requires for about 10 Seconds. IODINE ACETONE: ■ Preston & morrell have shown that the addition of a small concentration of iodine to acetone slows it’s rate of Decolorization without reducing its specificity. ■ With 0.35%iodine the exposure can be lengethed from about 2 seconds to 30 seconds or longer.
- 21. COUNTERSTAIN 1. Dilute carbol fuchsin: ■ This stain when applied gives the strongest red staining ■ However the colouration may be so dark that some gram negative bacteria mY be difficult to distinguish from gram positive ones. ■ It’s is better to use one of the following weaker counterstain. 1. BASIC FUCHSIN COUNTERSTAIN : It is applied for 10-30 sec this is recommended for general use. 2. NEUTRAL RED COUNTERSTAIN: applied for 2-4 min this is recommended for demonstrating gonococci and other intracellular gram negative bacteria. 3. SAFRANIN COUNTERSTAIN: Safranin 0.5 % in distilled water
- 22. Reporting Gram smears The report should include the following information: ■ Numbers of bacteria present, whether many, moderate, few, or scanty ■ Gram reaction of the bacteria, whether Gram-positive or Gram-negative ■ Morphology of the bacteria, whether cocci, diplococci, streptococci, rods, or coccobacilli. Also, whether the organisms are intracellular. ■ Presence and number of pus cells ■ Presence of yeast cells and epithelial cells.
- 23. USES OF GRAM STAIN: ■ To differentiate bacteria into gram positive & gram negative ■ For identification: gram staining from bacterial culture gives an idea to put the corresponding biochemical tests for further identification of bacteria. ■ To start empirical treatment: gram stain from the specimen gives a preliminary clue about the bacteria present ( based on the shape and gram staining property of the bacteria) so that the empirical treatment with broad spectrum antibiotics can be started early before the culture report is available. ■ For Fastidious organisms: such as Haemophilus which takes time to grow in culture, gram stain helps in early presumptive identification. ■ Yeasts: in addition to stain the bacteria, gram stain is useful for staining certain fungi such as Candida and Cryptococcus ( appear gram positive) ■ Quality of specimen: gram stain helps on screening the quality of the sputum specimen before processing it for culture. Presence of More pus cells and less epithelial cells indicates good quality specimen.
- 24. MODIFICATIONS OF GRAM STAINING: ■ KOPELOFF & BEERMAN’S MODIFICATION: Primary stain and counter stain used are methylviolet and basic fuchsin respectively. ■ JENSEN’S MODIFICATION: This method involves use of absolute alcohol as decolourizer and neutral red as counterstain It is useful for meningococci and gonococci. ■ BROWN &BRENN MODIFICATION: This is used for Actinomycetes.
- 25. LIMITATIONS ■ The sensitivity of the Gram stain procedure is low. Sometimes, you may fail to see the organism in Gram Stain smear, but the same clinical specimen may yield organisms when cultured. To be visible on a slide, organisms that stain by the Gram method must be present in about 104 to 105 organisms per milliliter of centrifuged fluid. ■ Gram staining technique is not recommended for spirochetes and mycobacteria. Mycobacteria stain weakly with gram stain, and bacteria such as Mycoplasma, Rickettsiae, Chlamydiae do not take up the dyes used in Gram stain or are too small to be seen with light microscopy. ■ Not all bacteria can be seen in the Gram stain. This is the list of medically important bacteria that can be not seen in Gram-stain.
- 26. Nearly all clinically important bacteria can be visualized using the Gram staining technique, the only exceptions being those organisms; • That exists almost exclusively within host cells (intracellular bacteria) (e.g., Chlamydia) • Those that lack a cell wall (e.g., Mycoplasma) • Those of insufficient dimensions to be resolved by light microscopy (e.g., spirochetes)
- 27. Name Reason Alternative Microscopic Approach Chlamydiae, including C. trachomatis Intracellular; very small Inclusion bodies in the cytoplasm Legionella pneumophila Poor uptake of red counterstain Prolong time of counterstain Mycoplasma pneumophila Poor uptake of red counterstain None Mycobacterium, includin g M. tuberculosis Too much lipid in cell wall so dye cannot penetrate Acid-fast stain Rickettsiae Intracellular; very small Giemsa or other tissue stains Treponema pallidum Too thin to see Dark-field microscopy or
- 28. QUALITY COUNTROL ■ Always check new batches of stain and reagents for correct staining reactions using a smear containing known Gram-positive and Gram- negative organisms. Variations in Gram Reaction ■ Various factors influence the results of Gram staining. Sometimes the result might be entirely different than you have anticipated. ■ Gram-positive bacteria may lose their ability to retain crystal violet and stain Gram negatively for the following reasons: – cell wall damage of bacteria due to antibiotic therapy or excessive heat fixation of the smear. – over- decolorization of the smear – use of an old Iodine solution that is yellow in color instead of brown (always store in a brown glass or other light opaque containers). – preparation of smears from old culture. A thick smear will require more decoloration than a thin smear
- 29. Organism Classic Presentation Variant Presentation Comments Streptococccus pneumoniae Gram-positive, lancet- shaped, diplococci Elongated cocci, resembling short bacilli May be misinterpreted as mixed organisms; over-decolorized cells may be mistaken for gram-negative coccobacilli. Acinetobacter spp. Gram-negative coccobacilli Gram-negative cocci; gram-variable staining is common May be mistaken for Neisseria spp. and reported as gram- negative cocci; search the smear to find some organisms that demonstrate elongated forms, which are not seen in Neisseria. Yeast, especially Cryptococc us neoformans Gram-positive round or oval cells with budding Gram-variable cells May be mistaken for artifacts; size and shape distinguish them from bacteria
- 30. Clostridium perfringens Boxcar- shaped gram- positive bacilli Gram-variable or Gram-negative bacilli Maybe mistaken for gram-negative bacilli; the boxcar shape is a clue that the organism is gram-positive; other Clostridia an d Bacillus spp. May also appear similar.
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