Microbiology Ch 2: Tools of the Lab

Inoculation

introduction of a sample into a container of media to produce a culture of observable growth

Incubation

inoculated media are placed in a temperature and atmosphere controlled environment (incubator) to promote growth

(****during the hours or days of this process, a culture develops as visible growth of microbes in the container of the medium

Isolation

if an individual bactria cell is separate from other cells and has space on a nutrient surface, it will grow into a mound of cells (= a colony ---> consists of ONE species)

Inspection

cultures are observed for the macroscopic appearance of growth characteristics

*cultures are examined under the microscope for basic details such as cell TYPE AND SHAPE

pure culture

grows only single known species of microorganisms

mixed culture

two or more identified species/microbes growing

contaminated culture

pure or mixed culture that has unwanted microbes growing

*this is important because it BAD and can induce FALSE DIAGNOSIS

ex) fungi.

information gathering

testing of cultures with procedures that analyze biochemical and enzyme characteristics, immunologic reactions, drug sensitivity, and genetic makeup

**IMPORTANT to gather the most information about your identification in order to rule out other microorganisms.

Identification

the goal of these procedures is to attach a name to the microbe, usually to the level of species.

-appearance

-biochemical tests

-genetic characteristics

-immunological testing

Magnification

the ability to make things look larger than they are

Resolving Power

ability to show detail

Refraction

the bending of light passing through convex surface of glass

total magnification

objective lens x ocular lens

Resolution (resolving power )

the capacity to distinguish or separate 2 adjacent objects

What is the purpose of oil?

to prevent light scattering for an overall clearer image

*used only 100X objective

Purpose of Staining

-increase contrast

- distinguish between gram-positive and gram-negative bacteria.

Positive staining

surfaces of microbes are negatively/positively charged and attract basic/acidic dyes

Negative Staining

microbe repels dye, the dye stains the background

Simple Stains

one dye is used; reveals shape, size, and arrangement

Differential Staining

use a primary stain and a counter stain to distinguish cell types

Example: gram-staim, acid-fast stain, endospore

Gram stain

-its a differential stain used to classify bacteria as gram-negative or gram-positive

-most significant technique in Microbiology

Physical states of media

1. liquid: nutrient broth; does not solidify

2. solid (agar): a firm surface for colony formation

3. semi-solid: clot-like consistency, contains agar solidifying agent

Types of Media

general, enrichment, selective, and differential media

General Media

used to grow a variety of bacteria, no special growth factors

Enriched Media

contains complex organic substances such as blood, serum, hemoglobin, or special growth factors required by microbes

examples:

- blood agar (microbes lice through)

- chocolate agar (already liced)

Selective Media

contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes

Differential media

allows growth of several types of microbes and displays visible differences among desired and undesired microbes

Endospore Stain

Used on clostridium and bacillus species

Green/Pink

What colors are endospores and vegetative cells after an endospore stain?

Flagellar stain

Binds to flagella, thickening & changing color

Pink/Blue

What colors are acid fast cells and non acid fast cells after an Acid fast stain?

Transmission electron microscope

Beam of electrons passes through specimen, excellent resolution of cellular details

Scanning electron microscope

Electrons pass over surface, produces 3-D images with great surface detail

2000x

Max magnification of light microscope

Magnification

Results from light or electron beam refracting as it passes through lens or magnetic field

Resolution

Ability to distinguish objects that are close together clearly

Contrast

Differences in intensity between 2 objects or an object and its background

1 um to 4+ mm

Protozoan size

3 to 10 um

Yeast size

200 nm to 750 um

Bacteria Size

20 to 400mm

Viruses

Synthetic/Defined Media

Same formula every time, every ingredient is known

Non-Synthetic/Complex Media

Contains extracts of animal, plant, or yeast products. Composition varies from batch-to-batch

Agar

Polysaccharide derived from Gelidium