1. Week 4W Review cultures: Exp 2B Lab Two & Exp 3 Lab Two
1 st
Review cultures: Exp 2B Lab Two & Exp 3 Lab Two
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Result Charts
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Exp 7. Prep of Smears
Exp 8. Simple Stain
Exp 10. Gram Stain
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4. Exp 7: Preparation of Bacterial Smears
Make sure slides are CLEAN!
On frosted side of slide, put organism’s initials and your own initials. Turn the slide over and draw a nickel-sized circle.
LCAT
P.A.
What we accomplish by this technique
Bacteria adhere to slide so they can be stained and observed.
Bacteria are killed, rendering pathogenic bacteria safe to handle.
An objective in preparing smears is to learn to recognize the correct density of bacteria to place on the slide.
Too many means light cannot pass through and it is difficult to visualize morphology of single cells
Too few, and they cannot be located on the slide.

5. Preparing Smears (cont.)
Turn the slide over (circle on the bottom)
Place a loopfull water in the center of the circle.
Using a sterile loop, aseptically transfer small amount of culture to the water—mix to emulsify.
Allow smear to air dry completely.
Once air dry, heat fix slide by quickly passing it 2-3 times over top of flame.
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. 
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.

6. Things that can go wrong when making a smear
Forget to clean the slide.
Use too much material - suspension should be just barely cloudy.
Use so much liquid that it takes forever to dry.
Heat the smear before letting it air dry, boiling the bacteria instead of attaching them.
Overheat the smear, melting cell walls and possibly breaking the slide

7. Week 4 Exp 7: Prep of Smears
Cultures: BS slant, ML slant, and EC slant
Procedure:
Prepare 6 slides (2 from each culture)
Follow directions on pg for cultures from solid mediums
Refer to Fig 7.2 for smear preparation

8. Exp 8: Simple Staining Material: Procedure: pg 60
Methylene blue stain
3 prepared slides from Exp 7- one of each culture
Procedure: pg 60
Put heat-fixed slide on staining tray at sink.
Flood smear with stain and leave on for 1-2 min.
Gently wash smear with water to remove excess stain.
Blot (DO NOT WIPE) with bibulous paper.
Examine organism under microscope under oil immersion.
Record observations as described on pg 63.
Simple staining is used to visualize bacteria on a slide
You can view basic shapes and arrangements of bacteria (refer to Fig 9.1 pg 65)
Shape: cocci, bacilli, spiral
Arrangement: singly, diplo-, tetrad, strepto-, staphylo-, vibrio-, spirilla, spirochete
All staining work is to be done at the sink
Care should be taken to work directly over the sink
Flood smear with stain (make sure it covers the entire area of the smear)
Rock or roll the slide to cover the area Place drop(s) of crystal violet stain on the smear ( 1 minute)

9. Exp 10: Gram Stain (refer to Fig 10.3 pg 72)
Procedure: pg 71
Primary stain: crystal violet; stains all cells purple (keep on 1min)
Wash off
Mordant: iodine; all cells stained purple (keep on 1min)
Decolorizing agent: alcohol; removes color from some cells (add drop by drop until alcohol has only slight blue tinge- not longer than 10 sec)
Immediately wash off
Counterstain: safranin; stains decolored cells pink (keep on 45 sec - 1 min)
Wash off blot dry with bibulous paper
Examine under oil immersion
Classifies bacteria into two large groups
Gram positive
Gram negative
All staining work is to be done at the sink
Care should be taken to work directly over the sink
Rock or roll the slide to cover the area
Different kinds of bacteria react differently to Gram stain—structural differences in their cell walls affect how they retain or lose the dye
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. 
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.

10. Things that can go wrong with a Gram stain
Make a bad smear to start with.
Apply stain to the wrong side of the slide.
Stain or rinse the smear incompletely.
Allow stain to dry up before rinsing.
Forget to decolorize or decolorize incompletely.
Rub off the material when you blot it.

11. Staphylococcus- Gram Positive11

12. Gram Negative Bacilli 12

13. Exp 10: GRAM STAIN TECHNIQUE
Smear, air dry, heat fix
Primary stain: CV (60 sec) - Rinse
Mordant: Iodine (60 sec) - Rinse
Decolorizing agent: 95% Alcohol (10 sec) - Rinse
Counter stain: Safranin (45-60 sec)
Week 4
If they have time to make more slides then required, they may make them. 5. Also, if students want to save slides that they may not have had a chance to view, we have small plastic cases that will hold up to 5 slides. 6. The students can put their name and section on a piece of tape and stick it to the case for future viewing. 7.The slide must be at least heat fixed to be saved or stained but no oil on it The immersion oil is not to be kept in their drawers. We have already had a spill in a drawer. Please put all bottles of immersion oil and also the stains back wear they are kept.
Rinse
Blot dry
- Observe 1000x oil immersion
Gram pos: blue/violet
Gram neg: pink/red
MLslant, BS slant, EC slant

14. The Microbe Library – A great Resource
Animation on Gram Stains
Typical Gram Stain