Microbiology Lab Practical Review Part 1

Compound Microscope

Light microscope/bright field microscope, is used to make the small, fine details of objects visible to the human eye

Lenses

pieces of glass with curved sides that concentrate or disperse light

Parfocal

they all have the same/similar focal planes (should not have to re-focus much between lenses)

Parcentric

an object centered in one lens will be centered in all lenses

Lenses are used for

Magnification and Resolution 

Magnification

enlargement of the object

Resolution

making the object clearer

To determine the total magnification 

multiply the magnification of the eyepiece with the objective

Eyepiece x Objective = Total Magnification

Resolution of objects (Resolution)

the ability of a lens to separate or distinguish between small objects that are close together. High resolution = clearer image

As you move from low power to medium power to high power

• Size of the field of view decreases

• Field of view is darker

• Size of the image increases

• Resolution decreases

• Working distance decreases

Refractive Index

a measure of how greatly a substance slows the velocity of light

• the direction of magnitude of bending are determined by the refractive indices of the 2 media forming the interface

Direct Staining

stains the cell directly since the dye is basic or positively charged and the cell wall is negatively charged (also called basic or positive staining)

Indirect Staining

stains the surroundings of the cell, the dye is negatively charged and is repulsed by the cell (also called acid or negative staining)

Hanging Drop Technique

allows us to observe the movement of microorganisms

• In comparison to wet mounts, there is more oxygen diffusion and less flowing

• It involves applying petroleum jelly to a coverslip, adding a culture drop, inverting it over the well, and flipping it to view the hanging drop under a microscope, providing better clarity and prolonged study of bacterial shape, arrangement, and movement.

Differential Stain

Distinguishes between Gram-positive and Gram-negative cells

Basic Dyes

• Same kind used for direct staining

• Crystal Violet

• Safranin

Gram-Positive

Cell walls have a thick peptidoglycan layer that retains the crytsal violet stain making it appear purple

Gram-Negative

cell walls have a very thin peptidoglycan later that does not retain crystal violet stain, allowing it to take up the safranin counterstain and appear pink/red

Gram Stain Process

1. Crystal violet (primary stain)

2. Iodine (mordant; fixes crystal violet)

3. Alcohol/acetone (decolorizer)

4. Safranin (counterstain)

Gram-positive bacteria

purple (thick peptidoglycan)

Gram-negative bacteria

pink (thin peptidoglycan + outer membrane)

Photoautotrophs

use CO2 for their carbon source (photosynthesis)

Photoheterotrophs

use organic carbon sources

Phototrophs

organisms that use light as an energy source

Conditions used to grow phototrophic bacteria

• Light

• Carbon source

• Oxygen requirements

• Temperature

• pH

• Specialized Media

• Incubation

Aerobe

require oxygen for growth

Anaerobe

growth is inhibited by O2

Facultative Anerobe

can grow in both aerobic and anaerobic conditions

Oxygen Requirements/Growth in Broth - Ways to create these conditions

Candle jar, Anaerobic Chamber, Gas Pack Jar, Thioglycolate broth

How do we describe things growing in liquid media?

Turbidity, Pellicle, and Sediment

Turbidity

cloudiness

Pellicle

mass of cells floating on the surface

Sediment

cells resting at the bottom

Selective Media

media that favors the growth of specific microorganisms by inhibiting others

• Ex. Media containing antibiotics

Differential Media

Media that distinguishes between groups of microorganisms based on differences in their growth and metabolic products

Important factors for media preparation

pH, sterility, proper temperature, agar concentration, nutrient composition

Endospores

form when the bacteria are stressed either through lack of nutreints or adverse environmental conditions

• The bacterium will replicate its DNA placing one inside the endospore

• Are resistant cells that are formed inside the cell

• Are not reproductive spores

Conditions for endospore formation

form only when bacteria experience stressful, unfavorable conditions. These stresses signal the cell that survival, not growth is the priority.

Endospores description

• Bacillus endospore - forming colonies

• They should look dry, grey, and spreading (some will have a wrinkly center)

How to identify Endospores in a stain and the stains used

1. Put 10 drops of water in a tube and add a loop of cells from your plate

2. Add 5 drops of Brilliant green dye in the tube and put in a boiling water bath for 10 minutes

3. Smear a slide, let it dry, heat fix it and stain with Safranin for 2 minutes

4. Rinse gently and dry before viewing

5. You should see green endospores in pink vegetative cells