Exercise 2 STUDYGUIDE

when do you use staining?

Staining can be used to make it easier to view
bacteria using a bright field microscope

What does staining do?

◦ Provides contrast (because bacteria is clear)
◦ Useful for identifying morphology, size, and arrangement of the bacterial sample

Different types of techniques to different:

◦ Simple
◦ Differential
◦ Structural

Types of dyes used in staining?

Cationic and Anionic

What are cationic dyes and what examples?

-Basic dyes and positively charged chromosomes

-Positively ion exhibits color

Examples: Methylene Blue or Crystal Violet

What are anionic dyes and what examples?

-acidic dyes and negatively charged chromosomes

-negative ion exhibits color

-repels by bacteria cell, so background takes on color.

Examples: Acid Fuchsin, Congo Red, Nigrosin

Negative Stain:

-Stain doesn’t penetrate the cell

-Negatively charged stain repelled by negative charge at cell surface

-Transparent cells against a colored background

what is negative staining good for?

-Good for examining morphology and size of bacterial cells
◦ Cells are not heat fixed, so distortion of the bacterial cells is minimized

-good for organisms that don’t stain easily.

Example of negative stain and what it does:

Nigrosin – black anionic dye (what we will be using)

◦ Repelled by the negatively charged bacteria cell surface

What is simple staining and what is it used for?

-One dye used to stain all cells present

-Useful in distinguishing different morphologies, arrangement, and relative sizes

-Colors the cells and provides contrast when using bright field microscopy

What is a simple stains charge and how is it used?

Positive charge of the dye interacts with the negative charge on bacterial cell wall
◦ Colors cell surface

What dye do we use for simple staining and is it cationic or anionic?

We use crystal violet which is cationic.

When making a smear prep from a broth to slide how do you do it?

-Aseptically transfer 1 loopful of culture to a
clean slide and spread to maximum thinness
-Allow to dry on slide dryer
-Heat fix by holding over microincinerator for
10 seconds - USE CLOTHESPIN!!

When making a smear prep from slant/plate to slide how do you do it?

-Add 1 loopful of water to the slide FIRST
-Aseptically collect bacterial sample from plate
or slant
◦ Just touch the loop to the bacterial growth (only
need small amount) on the plate or slant, mix
well to suspend
-Allow to dry on slide dryer
-Heat fix by holding over microincinerator for
10 seconds - USE CLOTHESPIN!!

Main difference with making a smear prep from a broth and a slant/plate?

If you have a broth don’t add loopful of water first, if you have a slant/plate add a loopful of water to the slide first.

Why do you want to spread so thin on a smear prep?

Aids in drying and in viewing later (thin layer of cells)

MICROSCOPE STUFF:

How to carry a microscope:

◦ Always carry microscope with two hands
◦ One hand should hold the handle and the other should support the base

How to store the Microscope correctly?

◦ Clean oil off 100x objective using lens cleaner and kimtech wipe
◦ Lower stage as low as possible
◦ Move the 4X objective into position
◦ Make sure cord is properly wrapped up

Microscopy is based on three principals:

1. Illumination

2. Magnification

3. Resolution

1. Illumination

◦ Light source (bulb)
◦ Condensor: directs light towards the objective lens in bright field microscopy (directs)
◦ Iris diaphragm: adjusts the diameter of the cone of light so that it just fills the objective lens (adjusts)

2. Magnification

◦ Ocular: 10X (start with)
◦ Objectives: 4X, 10X, 40X and 100X

Total magnification formula:

ocular x objective = total magnification

3. Resolution

◦ Resolution is the smallest distance between two objects which can be seen as separate

◦ Our scopes resolving power is ~0.2μm while the average microbe is 1.0μm

Oil Immersion: What objective and how to use?

-100x

-MUST use oil (lens must touch the oil)

-Oil has the same refractive index as glass

-Oil stops refraction of light between specimen and lens

When done with oil immersion, how to clean?

When done, clean oil off oil immersion lens with lens paper and cleaner fluid!

resolution/resolving power formula and meanings:

d (resolution) = wavelength (lambda)/2NA

-NA= Numerical aperture: light gathering capacity of the lens (depends on lens using)

A smaller d value (resolution value) means

better resolution

MORPHOLOGY OF BACTERIA:

Is staining with conjunction with microscopy used for determining morphology?

YES!

Bacteria have many different shapes:

What is a circle bacteria?

Cocci

What is two circular connected bacteria?

Diplococci

What is a long line (chain) of circular bacteria?

Streptococci

What are grape like clusters of circular bacteria?

Staphylococci

What are four circular bacteria shaped like a square called?

tetrads

What is a rod shaped bacteria?

Bacilli

What is a long line (chain) of rod shaped bacteria?

Streptobacilli

What is a circular and rod like bacteria called?

Coccobacilli

What is a comma shaped bacteria called?

Vibrio

What is a corkscrew/spiral rigid shaped bacteria called?

Spirochete

What is the bacteria shape called where all of them are different in shape and sizes under the same conditions?

Pleomorphic