CHAPTER 3 OBSERVING MICROORGANISMS THROUGH A MICROSCOPE I. UNITS OF MEASUREMENT - See Table 3.1 in text. + Fig. 3.2 II. MICROSCOPY: THE INSTRUMENTS A. COMPOUND LIGHT MICROSCOPY Figure 3.3 1. Have ocular and objective lenses 2. Calculate total magnification by multiplying the objective x the ocular (10x). a. Objective lenses are 10x (low power), 40x (high power), and 100x (oil immersion) 3. Resolution ability to distinguish between points a. White light can go to about 0.2 µm 4. Brightfield is used for stained smears; unstained use darkfield or phase-contrast (most common). a. Treponema pallidum B. FLUORESCENCE MICROSCOPY 1. Principle - substances absorb short wavelengths (ultraviolet) and give off light at a longer wavelength. 2. Need a microscope with special filters. 3. Good for screening where there are few organisms. a. Auramine O - a fluorochrome for Mycobacterium tuberculosis. Microbiology 8 CHAPTER3taft 1
C. ELECTRON MICROSCOPY 1. Uses a beam of electron instead of light. 2. Get better resolution and can see organisms 100,000 times smaller than with visible light. a. Have to use to see viruses. III. PREPARATION OF SPECIMENS FOR LIGHT MICROSCOPY A. PREPARING SMEARS FOR STAINING 1. Preparing Smears a. Have to fix specimen to slide. See lab book for details. Use heat to attach specimen to slide. Will use heat plate in our lab. Can also use methyl alcohol. Get better preservation of specimens. b. Stains are salts composed of positive and negative ions one of which is colored. Basic dyes = positive colored ion. Acidic dyes = negative colored ion. Bacteria are slightly neg. So basic dyes are attracted to bacteria. These are crystal violet and safranin in gram stain. 2. Simple Stains a. Solution of a single basic dye. b. Mordant Added to stain to intensify. c. Methylene Blue - Used in lab. Microbiology 8 CHAPTER3taft 2
3. Differential Stains - Act differently with different bacteria and used to distinguish among them. a. Gram Stain - Very Important Tool fig. 3.10 What s Happening? - Gram positive bacteria retain the purple/blue stain (crystal violet + mordant Iodine) after the decolorization step; gram-negative bacteria do not and thus appear pink/red from the counterstain (safranin). Why? - Due to structural differences in cell wall. b. Acid-Fast Staining - Called AFB Stain - Binds strongly to bacteria that have waxy material in cell wall. There is a quick stain available, doesn t require heat. AFB, Mycobacterium tuberculosis and Mycobacterium avium, and Norcardia retain carbolfuchsin after acid-alcohol decolorization and appear red; non acid-fast take up the methylene blue counterstain and appear blue. Fig 3.11 4. Special Stains - Stains used to show structures such as capsules, flagella, or endospores. a. Capsule stain- Colloidal soln of dark particles. Ex. Use India Ink for Cryptococcus neoformans. Shows capsule. Fig. 3.12 a b. Endospore- malachite green as primary and safranin as secondary. Clostridium tetani, Fig 3.12b Microbiology 8 CHAPTER3taft 3
III. Slide Show c. Flagella- use mordant to increase diameter so can view white light. Fig. 3.12c Microbiology 8 CHAPTER3taft 4
GRAM STAIN PROCEDURE 1. The slide should be heat fixed on the heat block, or methanol fixed, prior to staining. 2. Methanol fixation is accomplished by flooding the slide with 95% methanol. Let the slide sit for 2 minutes, drain off the excess methanol, and allow to air dry. 3. Flood the slide with crystal violet for 30 seconds. Rinse with water. 4. Flood the slide with iodine for 60 seconds. Rinse with water. 5. Flood the slide with decolorizer for about ten seconds decolorization is complete when the solution runs clear from the slide. Rinse with water. 6. Flood the slide with safranin for 30 seconds. Rinse with water. 7. Blot the slide dry with absorbent paper and examine the slide under an oil immersion lens. INTERPRETATION gram negative organisms = pink color gram positive organisms = blue color Microbiology 8 CHAPTER3taft 5