GRAM STAINING AND its MODIFICATIONs.pptx

gram staining and its modification

1. GRAM STAINING AND MODIFICATION Presented By Dr Mohan Singh Dhakad PG 1 st Yr Department Of Microbiology

2. CONTENT Introduction Procedure Interpretation Mechanism Uses Modification Summary Reference

3. GRAM STAIN Most widely used staining method. Developed by Christian Gram in 1884.

4. PREPARATION OF SMEAR Labeling of slide : With diamond pencil. Preparation of smear : Smear should be prepare evenly covering an area of about 15-20 mm diameter on the slide. From fluid specimen : Take loopfull specimen with sterile inoculating wire and spread on slide. From csf specimen : Centrifuge(>1ml) and then make smear From swab : Roll the swab on slide From culture plate : Put a drop of saline on a clean slide with the help of sterile inoculating wire ,individual colony is picked up and mix with normal saline.

5. Procedure of staining

6. INTERPRETATION Gram + ve bacteria resist decolorization and retain color of primary stain , violet Gram – ve bacteria decolorized and take counter stain and appear , pink

7. MECHANISM/PRINCIPLE PH theory : Gm + ve bacteria have more acidic cytoplasm so can retain basic dye for longer time. Dye- Iodine complex which retain inside the cell Cell wall theory : Gm + ve bacteria have thick peptidoglycan layer with tight cross linking, Alcohol also shrink pores of thick peptidoglycan layer so large dye iodine complex are not able to penetrate .

8. USES OF STAIN To differentiate bacteria into gram + ve & gram – ve For identification of bacteria from culture with corresponding biochemical test To start empirical treatment For staining of certain fungi: Candida,Cryptococcus Quality of sputum specimen Preliminary clue to perform anaerobic culture ex clostridium do not grow in routine culture

9. Original Method Of Gram Stain Original method of gram stain (1884) used: Primary Stain = Anilline -gentian violet Mordant = Lugol’s Iodine Decolourizer = Absolute Alcohol Counterstain = Bismarck Brown

10. MODIFICATION OF GRAM’S STAIN Kopeloff and Beerman's Modification Jensen's Modification - Gonococci and meningococci Preston and Morrell's Modification Quick Gram Method For Single Slide Gram Method For Multiple Slide Brown and Brenn Modification : Used For Actinomycetes .

11. PREPARATION OF STAIN Crystal Violet/ Methyl Violet: 1. Crystal Violet/ Methyl Violet 10 Gm 2. 100% Ethanol(absolute Alcohol) 100ml 3. Distilled Water 1ltr Crystal/methyl violet is used at concentration of 0.5-2% solution. Gram positive staining can be strengthened by addition of sodium bicarbonate or ammonium oxalate

12. Kopeloff & Beerman Stain Solution A - Methyl violet 10g -Distilled water 1 L Solution B -Sodium bicarbonate 50g -Distilled water 1 L Shortly before use, mix 30 volumes of sol. A to 8 volumes of sol. B. This mixture precipitate within few days so can't store for long time.

13. Preston & Morrell’s stain Crystal violet 20g Methylated spirit 200ml Ammonium oxalate, 1% in water 800ml

14. IODINE SOLUTION Gram’s ( Lugol’s )iodine solution: 1. Iodine 10gm 2. Potassium iodide 20gm 3. distilled water 1000ml Weak but economically equal effective . Kopeloff & Beerman’s iodine : 1. Iodine 20gm 2. Sod.Hydroxide 1mol/L 100ml 3.Distilled water 900ml Strong and more alkaline

15. DECOLORIZERS Fastest - acetone(2-3 sec) Useful when only single slide is to be stained. However, short period of exposure is difficult to control when many slide has to be stain. Slowest - 100% ethanol (1 min or more) This act more slowly. So it should be applied and re-applied for about 1 min. Intermediate- acetone-alcohol(10 sec.) Ethanol, 95% 100ml Acetone 100ml

16. Iodine-acetone :Preston & Morrell have shown that addition of a small concentration of iodine to acetone slows the rate of decolourization without reducing its specificity. The decolourizer made are as follows: Liquor Iodi Fortis 1. Iodine 10gm 2. Potassium iodide 6gm 3. Methyleted spirit 90ml 4. Distilled water 10ml

17. Iodine-acetone Liquor iodi fortis 35ml Acetone 965ml Hazard :irritating aerosol may be produced Weak iodine-acetone : .035% iodine Good gram differentiation obtained and aerosol irritation may also be avoided

18. COUNTER STAINS Dilute carbol fuchsin: Strongest red staining within 10-30 sec. Coloration is so dark that some time difficult to distinguish Gram negative from Gram positive bacteria. Basic fuchsin : Applied for 10-30 sec. This is recommended for general use. Basic fuchsin 0.5 gm Distilled water 1000ml Neutral red : gonococci & meningococci Safranine : Used as 0.5% in distilled water.

19. MODIFICATION OF GRAM’S STAIN KOPELOFF AND BEERMAN'S MODIFICATION :- Primary stain: solution of freshly prepare methyl violet with sodium bicarbonate in distilled water. Mordant : iodine dissolved in 4% NaOH solution. Decolonization: acetone Counter Stain: Basic fuchsin

20. Methyl violet stain× 5 min. Iodine solution to wash away all the methyl violet and crystalline dye-iodine precipitate. ↓ Iodine solution× 2 min. ↓ Decolourized with acetone ×2-3 seconds. ↓ Wash with tap water ↓ Basic fuchsin×30 seconds.. ↓ Wash with tap water about 5 seconds , Procedure Kopeloff And Beerman's Modification

21. Jensen's Modification For Neisseria species Methyl violet as primary stain, Iodine and potassium iodide in water as mordant, Absolute alcohol as decolourizer Neutral red as counter stain.

22. Procedure of Jensen's Gram method D ry and fix the smear and place on rack. ↓ Methyl violet ×30 seconds. ↓ Tilt the slide, pour Lugol's (1%)iodine to wash away the stain, ↓ Cover with fresh iodine 30 seconds. ↓ Tilt the slide and wash off the iodine with95% or 100% alcohol (ethanol). ↓

23. Cont. Treat with fresh alcohol. until colour ceases to come out of the smear. This will take 30 seconds or longer ↓ Wash with tap water ↓ Pour on neutral red (0.1%) counterstain ×2 minut ↓ Wash with water and blot dry

24. Procedure of Preston And Morrell's Modification The primary stain used in this modification is ammonium oxalate-crystal violet (30 sec). ↓ The smear is washed in Lugol's iodine and further treated with iodine solution(30 sec). ↓ The smear is decolorized using iodine-acetone decolourizer (30 sec) ↓ wash with water

25. Cont. Counterstained using dilute carbol fuchsin solution(30 sec). ↓ Wash with tap water This method has been further modified to overcome the irritating iodine in aerosols by reducing the iodine concentration to one-tenth and shortening the duration of decolonization to > 10 seconds

26. Quick Gram method for single slides Flood the slide with crystal or methyl violet stain ×5 seconds. ↓ Tip off the stain and flood the tilted slide with iodine solution ×5 seconds. ↓ Tip off the iodine and flood the tilted slide with acetone ×2 seconds ↓ Flood the slide with basic fuchsin counter stain×5 seconds. ↓ Wash off with water, blot and dry.

27. Gram method for multiple slide Slowly acting decolorizer such as iodine-acetone. Mark the slide with a number identifying the smear. ↓ Make, dry and heat-fix the smears. ↓ Cover slides with methyl violet stain and leave to act for at least 30 seconds. ↓ Wash off the stain with a flow of water directed at it through the tubing from the tap. ↓

28. Cover with Lugol's 1% iodine, displacing the residual water from the pre-ceding wash. ↓ Leave the iodine to act for at least30 seconds. ↓ Wash off the iodine with water. ↓ Cover each slide iodine acetone × 30 sec ↓ Basic fuchsin counter stain ×30 sec ↓ Wash with water

29. summary Original method of Gram staining Kopeloff & Beerman Jensen Preston &Morrell’s 1° stain Anilline -gentian violet Methyl violet,(sod bicarb )x 5 mnt Methyl violet x 30 sec Crystal violet(ammo oxal ) x30 sec Mordant Lugol’s Iodine I (NAOH) x2 mint Lugol’s Iodine x30 sec Lugol’s Iodine x 30 sec Decolourizer Absolute Alcohol Acetone 2-3 sec absolute alcohol x 1 mnt iodine-acetone x 30 sec Water wash Counter stain Bismarck Brown Basic fuchsin x30 sec Neutral red 2 mnt Dilute carbol fuchsin x30 sec Water wash

30. Reference Mackie & McCartney practical medical microbiology 14 th edition Indian Council Of Medical Research Essentials of medical microbiology Apurba S Sastry , 3 rd edition