1. Staining Microbiology Professor Sidelsky 2007

2. Part Two - Staining • How to Make a Smear • Stain with Simple Stain • Stain with Gram Stain • Acid Fast • Specialized Staining( Endospore and Capsule) • Negative Staining

3. How to Make a Smear • 1.  Label the frosted side of your slide with your initials, the name of the organism, and the date.  • Turn the slide over and draw an oval on the reverse side with a Sharpie • 2. You are going to make the smear on the frosted side

4. Broth Directions for making a smear • *****If you are using broth follow these directions • Flame your inoculating loop. • Use aseptic technique and remove the top of the culture tube, flame the mouth or the culture tube, and dip the loop into the broth.  Make sure that the loop is filled. • Transfer the loopful of broth and bacteria to the slide. Using a circular motion, spread the broth on the slide. • This is now a " smear" • Allow the smear to dry • When the smear has been allowed to " air dry" , pass the smear through the flame to " heat fix" - Heat fixation causes the proteins and cell parts to coagulate and stick to the slide. • Let the slide cool.

5. How to Make a Smear- Plate or Slant • *****If you are using colonies from a plate( Petri Dish) • Place a drop of water on the slide. • Using aseptic technique, " pick" a colony from the Plate in order to study the cells that make up a colony • If you are using a slant • Using a circular motion spread move the inoculating loop in a circular motion in the drop of water. Start in the center of the drop and move in a circular motion to the outside of the drop.  The objective is to have fewer cells on the outside of the circle.  • Follow the directions from above • Don't forget to " air dry" and to " heat fix" before staining

6. Simple Stain • Simple Stains- Crystal violet and methylene blue • 1.  Place a drop(s) of stain over the smear. Make sure it covers the entire area of the smear.  Leave the stain on the smear for one minute.  Rinse with water from the bottle.  • 2. Refer to page 64 for cellular morphology.

7. The Gram Stain • Gram Stain- See Gram Stain Directions on separate page.  Please refer to pages 71-73 in your laboratory manual. • All staining work is to be done at the sink • Care should be taken to work directly over the sink • Place drop(s) of crystal violet stain on the smear ( 1 minute) • Rock or roll the slide to cover the area • Use the water bottle to drip water down the slide

8. Gram Stain ( II) • Place drop(s) of iodine on the slide ( 1 minute) • Place drops of alcohol on the slide 10 seconds ( KEY – do not leave on longer than 10 seconds or it will decolorize) • Place drop(s) of saffranin on the slide for 1 minute • Rinse with water from the bottle • Let the slide air dry

9. The Microbe Library – A great Resource • Animation on Gram Stains http://www.microbelibrary.org/ASMOnly/details.asp?id=2020&Lang=

10. Gram Stain( step by step) • http://medic.med.uth.tmc.edu/path/grampro.htm • http://www.lumen.luc.edu/lumen/DeptWebs/microbio/med/gram/tech.htm

11. Typical Gram Stain http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/Gram3.htm http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/Gramstains/small/tocframeset1.htm

12. Staphylococcus

13. Gram Negative bacteria

14. Gram Stain animations and information • http://www.microbiologybytes.com/video/Gram.html

15. Bacterial Cell Morphology

16. Acid Fast Stains( Mycobacterium) Acid- Fast Stain • Carbol Fuschin Stain is a red stain that is soluble in a lipid environment. The outer cell wall of the mycobacterium is mycolic acid • The stain can be absorbed by placing the stain over a beaker boiling water. Heat assists the stain inits penetration of the waxy cell wall • Cool and rinse with cool water • Add acid- Alcohol drop by drop until the alcohol runs alost clear. • Rinse with water • Counterstain by applying methylene blue for at least two minutes

17. Acid Fast Staining • A shows cells that are stained with methylene blue – These re non acid fast • B shows cells that are stained with carbol fuschin which is retained in the lipid cell walls

18. Acid Fast

19. Differential Staining( Spores) • Add Malachite Green to slide and steam over a beaker of boiling water ( beaker may be placed on a hot plate) If stain starts to evaporate, add additional stain. Stain should remain on for 2-3 minutes • Remove slides from hot plate and cool Rinse with water • Counter stain with Saffranin for a minute. Rinse with water

20. Endospore structure

21. Endospore

22. Capsule stain • Crystal violet is applied to the bacterial smears • The decolorizing agent is Copper sulfate( 20%) which washes the primary stain out of the capsule without removing the stain from the cell wall

23. Capsule stain( Klebsiella pneumoniae and Streptococcus pneumoniae)

24. Negative staining • Place a drop of nigrosin on the end of a slide- Use a plain slide • Use the inoculating loop to mix some of the bacterial culture with the drop of stain • Use a clean slide. Hold at a 45o angle against the drop. The drop will spread along this line • Push the slide away from the previously spread drop

25. Negative staining

26. Negative staining