PowerPoint to accompany Foundations in Microbiology Fifth Edition Talaro Chapter 3 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Tools of the Laboratory: The Methods for Studying Microorganisms Chapter 3

The 5 I’s of culturing microbes

1. I

noculation – introduction of a sample into a container of media

2. I

ncubation – under conditions that allow growth

3. I

solation –separating one species from another

4. I

nspection

5. I

dentification 3

Fig. 3.1a

Fig. 3.1b

Fig. 3.1c

Fig. 3.1d

Isolation • If an individual bacterial cell is separated from other cells & has space on a nutrient surface, it will grow into a mound of cells- a

colony

• A colony consists of one species 8

Isolation technique 9

Fig. 3.3

Media – providing nutrients in the laboratory • • Most commonly used: – –

nutrient broth

& peptone – liquid medium containing beef extract

nutrient agar

peptone & agar – solid media containing beef extract,

agar

is a complex polysaccharide isolated from red algae – solid at room temp, liquefies at boiling (100

o

C), does not resolidify until it cools to 42

o

C – provides framework to hold moisture & nutrients – not digestible for most microbes 11

Types of media • •

synthetic

– contains pure organic & inorganic compounds in an exact chemical formula

complex or nonsynthetic

– contains at least one ingredient that is not chemically definable • •

general purpose media

- grows a broad range of microbes, usually nonsynthetic

enriched media

- contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes 12

Enriched media Blood agar plate Bacteria from human throat Grows streptococci and other pathogens Chocolate agar plate Grows

Neisseria

(causes gonorrhea) 13

• •

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 14

Selective & Differential Media 15

Selective & Differential Media

Mannitol salt agar-

selectively grow

Staphylococcus

species. Contains phenol Red that changes color with pH change.

Contains mannitol, a sugar that is Converted to acid.

S. aureus

uses mannitol (yellow) NaCl also inhibits salt-sensitive species

MacConkey agar-

differentiates between Lactose-fermenting bacteria (colony center red) and lactose-negative bacteria (off-white).

Isolation of gram-negative enterics 16

Differential media 17

Miscellaneous media • •

reducing medium

– contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria

carbohydrate fermentation medium

contains sugars that can be fermented, converted to acids, and a pH indicator to show the reaction; basis for identifying bacteria and fungi 18

Carbohydrate fermentation media 20

Key Characteristics of the Microscope • •

magnification

– ability to enlarge objects

resolving power

– ability to show detail 21

compound light microscope 22

Pathway of light 23

Effect of wavelength on resolution 24

Oil immersion lens 25

Effect of magnification with oil immersion 26

Types of light microscopes • • •

Bright-field

– most widely used, specimen is darker than surrounding field

Dark-field

– brightly illuminated specimens surrounded by dark field, uses stop condenser

Phase-contrast

– transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures 27

3 views of a

Paramecium

Bright-field Dark-field Phase-contrast 28

Fluorescence Microscope • Modified compound microscope with an ultraviolet radiation source and a filter that protects the viewer’s eye • Uses dyes that emit visible light when bombarded with shorter uv rays.

• Useful in diagnosing infections 29

30

Electron microscopy • Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds.

• Electron waves are 100,000X shorter than the waves of visible light.

• Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength.

• Magnification between 5,000X and 1,000,000X 31

32

2 types of electron microscopes • •

Transmission electron microscopes

(TEM) – transmits electrons through the specimen; darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts

Scanning electron microscopes

(SEM)– provides detailed three-dimensional view. SEM bombards surface of a whole,

metal-coated specimen

with electrons while scanning back and forth over it.

33

Transmission Electron Micrograph 34

Scanning Electron Micrograph 35

Specimen preparation • •

wet mounts & hanging drop mounts

– allow examination of characteristics of live cells: motility, shape, & arrangement

fixed mounts

are made by drying & heating a film of specimen. This

smear

is stained using dyes to permit visualization of cells or cell parts.

36

Staining • •

cationic dyes

- basic, with positive charges on the chromophore

anionic dyes

- acidic, with negative charges on the chromophore • • surfaces of microbes are negatively charged and attract basic dyes –

positive staining

.

negative staining

– microbe repels dye & it stains the background 37

Staining • •

simple stains

–one dye is used

differential stains

– use a primary stain and a counterstain to distinguish cell types or parts. examples: Gram stain, acid-fast stain and endospore stain •

special stains

: capsule and flagellar stains 39

Types of stains 40