Week 4W Review cultures: Exp 2B Lab Two & Exp 3 Lab Two 1 st Review cultures: Exp 2B Lab Two & Exp 3 Lab Two Update Journals Result Charts NEW Exp 7. Prep of Smears Exp 8. Simple Stain Exp 10. Gram Stain 9/10

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.

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.

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

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 54 for cultures from solid mediums Refer to Fig 7.2 for smear preparation

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)

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.

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.

Staphylococcus- Gram Positive 11

Gram Negative Bacilli 12

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. 8. 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

The Microbe Library – A great Resource Animation on Gram Stains http://www.microbelibrary.org/ASMOnly/details.asp?id=2020&Lang= Typical Gram Stain http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/Gram3.htm