1. GRAM STAINING

2. OBJECTIVES ● Describe reagents used in Gram stain & purpose of these reagents. ● Color expected of Gram Pos & Gram Neg after performing the procedure. ● Explain reason of differential stain by Gram Pos & Gram Neg

3. REAGENTS USED. 1. Gram Crystal Violet 0.5% 2. Gram Iodine. a) Potassium Iodide 2% b) Resublimed Iodine 1% 3) Gram Decolorizer a)Methanol 80% b)Acetone 20% 4.Gram Safranine Gram negative Gram positive

4. Cocci Bacilli

5. Reagents Used in Gram Staining 1.CRYSTAL VIOLET ● Primary stain ● Violet colored, stains all micro-org. 2 GRAM IODINE ● Mordant ● Forms Crystal violet iodine complexes

6. Cont. 3. DECOLORIZER Acetone + Methanol Removes Crystal violet iodine complex from thin peptidoglycan layers Dissolves outer layer of Gram negative org

7. Cont 4.GRAM SAFRANINE ● Counter stain ● Red colored ● Stains thin walled Gram neg org ● Pus cells cytoplasm & lobes of nuclie also stain red

8. The Gram Stain Procedure Step 1 - Prepare a Smear ● Suspend some of the material to be stained in adrop of water on a microscope slide, ● spread the drop to about the size of a nickel. ● Allow to air dry. ● Heat fix by gently warming.

9. Step 2 Step 2 - Apply the Primary Stain Flood the Smear with Crystal Violet Allow to stand for 1 min Rinse with water to remove excess stain

10. Step 3 and 4 Step 3 - Apply the Mordant ● Flood the Smear with Iodine solution ● Allow to stand 2 min Step 4 - Rinse Rinse with water to remove excess Iodine

11. Step 5 - Decolorize and Step 6. ● Drip Decolorizer (80% Methanol +20% Acetone) across the slide about 5 sec ● The effluent should appear pale or clear. Step 6 - Rinse ● Rinse with water to remove excess alcohol

12. Step 7 - Counterstain ● Flood the slide with Safranin solution ● Let stand for 2 minutes Step 8 - Rinse, Dry and Observe ● Rinse with water to remove excess stain ● Blot dry ● Observe under Oil Immersion

13. Gram Staining Video (5 min) https://www.youtube.com/watch?v=9Bnak_ITqck&feature=player_embedded

14. PLATE STREAKING https://www.youtube.com/watch?feature=player _detailpage&v=Ay2hhujTuvg

15. Pouring Agar https://www.youtube.com/watch?feature=player_detailp age&v=OljTdYH_Wtg https://www.youtube.com/watch?feature=player_detailpage&v=X4L16Vu5MSk

16. Three Sector Streak (t streak): ● Sterilize the wire loop. ● Cool the loop by touching it on the edge of the sterile agar plate. ● Dip the loop into the broth culture containing the mixture of bacteria. ● Lift the lid of the plate just enough to insert the loop. Drag the loop over the surface of the top one-third of the plate back and forth in a "zig-zag" formation. ● The loop has picked up thousands of bacteria which are spread out over the surface of the agar.

17. Cont ● Sterilize the loop in the flame. ● Turn the plate 90 degrees and drag the loop through the area you have just streaked two to three times and continue to drag the loop in a "zig-zag" formation in the remaining half of the plate without touching that area again. ● Sterilize the loop in the flame. ● Turn the plate 90 degrees. Repeat the procedure. Drag the loop two to three times through the area you just streaked, and fill in the remaining area of the plate (zig-zag formation), being very careful not to touch any of the areas you previously streaked.

18. CONT ● Incubate the plate for 24 hours. If you streaked correctly, you ● will see isolated colonies in the third sector. ● The heaviest growth will be in the first sector. There will be less growth and some isolated colonies in the second sector. The third area should have the least growth with isolated colonies.

19. MEDIA ● Selective, elective and diagnostic media (different agars) – students to research growth characteristics; and prepare plates of each type of media. ● Consider growth characteristics for:- E. coli Staphylococcus aureus Enterococcus spp

20. SELECTIVE MEDIA

22. Selective Bacterial Growth Media ● Selective media contain ingredients that inhibit the growth of certain types of bacteria and/or encourage the growth of others. ● This type of media is useful in helping to identify unknown bacteria and in encouraging the growth of only the types of bacteria that the microbiologist is interested in cultivating.

23. For example MacConkey’s Agar (MAC) is used to cultivate Gram-negative bacteria, by discouraging the growth of Gram positive bacteria through the use of crystal violet dyes and bile salts.MacConkey’s Agar Another selective medium, Mannitol Salt Agar (MSA), has a high concentration of sodium chloride, which selects for halophiles (salt-loving bacteria) such as members of the genus Staphylococcus.Mannitol Salt Agar

24. Contains lactose as substrate and Neutral red as an indicator. Bacteria fermenting lactose produce acid and this changes the colour of the indicator and the colonies turn pink. Use: To differentiate lactose fermenters (E. coli, Klebsiella) from non-lactose fermenters (Salmonella,Shigella) Mac Conkey Agar

26. SELECTIVE MEDIA ● MacConkey is a seletive Media but also Differential Media. ● It contains PH Indicator and sugar lactose. ● Gram -ve bacteria are able to ferment lactose ● Crysatl violet in this will stop the gram +ve gram. ● Manitol Salt Agar.

27. DIFFERENTIAL MEDIA ● This is used to differntiate closely related organisms or groups of organisms. ● Eg MacConkey agar ● Methylene Blue agar : Inhibits the growth of most Gram +ve. ● Manitol Salt Agar: used to isolate Staphylococci

28. Nutrient Media. All elements that most bacteria need for growth they are non selective. eg. Plate count agar Nutrient Agar Trypticase Soy agar

29. MacConkey Eschrchia Coli : Pink Colonies Proteus Mirabili : Colourless Colonies Salmonella Tyohimurium: Colourless Colonies. Staphylococcus Aureus: No growth.

30. E. Coli Grow on MacConkey Gram negative. Apperas in singles or pairs Reduce nitrate Oxidase negative Produce brighr pink lactose fermentos.