microbiology chapter 2

The Five I's of Microbiology

1. Inoculation

2. Incubation

3. Isolation

4. Inspection

5. Identification

Inoculation

the introduction of an inoculum into media to culture microbes

Culture

to grow microorganisms

Medium (plural media)

nutrients for the growth of microbes

Inoculum

a small sample of microbes

Incubation

Incubator: a temperature-controlled chamber to encourage the multiplication of microbes•

Typical temperatures used in laboratory propagation of microorganisms: 20 to 45°C•

Medically-significant organisms grow 35-37 °C for 18-24 hr•

Atmospheric gases such as oxygen or carbon dioxide maybe required for the growth of certain microbes•

- During the incubation period, the microbe multiplies and produces growth that is observable macroscopically

Various Conditions of Cultures

pure culture, mixed culture, contaminated culture

Physical states of media

-liquid

-semisolid

-solid (can be converted to liquid)

-solid (cannot be liquefied)

Agar

Complex polysaccharide isolated from Gelidium• Solid at room temperature•

Liquefies at 100°C; solidifies at 42°C•

Flexible and moldable•

Not a digestible nutrient for most microorganisms

general purpose media

Grow as broad a spectrum of microbes as possible• Generally complex

enriched media

Contains complex organic substances such as blood, serum, hemoglobin, or special growth factors for the growth of fastidious microbes•

- Used in the clinical laboratory to encourage growth of pathogens present in low numbers

selective media

Contains one or more agents that inhibit the growth of a certain microbe or microbes:•

- Important in the primary isolation of a specific type of microorganism from a sample containing dozens of different species•

- Speed up isolation by suppressing unwanted background organisms and favoring the growth of the desired ones

differential media

Allow multiple types of organisms to grow but display visible differences in how they grow:•

Variations in colony size or color•

Media color changes•

Production of gas bubbles•

Variations often come from chemicals in the media with which microbes react

Transport media

used to maintain and preserve specimens that have to be held for a period of time before clinical analysis

Carbohydrate fermentation media

Contains sugars that can be fermented with a pH indicator to show this reaction.

Isolation

Based on the concept that if an individual bacterial cell is separated from other cells on a nutrient surface, it will produce a discrete mound of cells called a colony

requires:

A medium with a firm surface•

A Petri dish•

An inoculating loop (streak plate method)

colony

a macroscopic cluster of cells appearing on a solid medium arising from the multiplication of a single cell

Inspection and Identification

Microbes can be identified through

- microscopic appearance

- characterization of cellular metabolism

- determination of products given off during growth, presence of enzymes, and mechanisms for deriving energy

- genetic and immunological characteristics

bright field microscopy

The most widely used type of light microscope

• Forms its image when light is transmitted through the specimen

• Can be used for both live, unstained material and preserved, stained material

dark field microscopy

A bright-field microscope can be adapted as a dark-field microscope by adding a special disc called a stop to the condenser

• The resulting image is a particularly striking one: brightly illuminated specimens surrounded by a dark (black) field

flourescence microscopy

The fluorescence microscope is a specially modified compound microscope furnished with an ultraviolet (UV) radiation source

• The name comes from the use of certain dyes (acridine, fluorescein) and minerals that are fluorescence. The dyes emit visible light when bombarded by short ultraviolet rays.

• For an image to be formed, the specimen must first be coated or placed in contact with a source of fluorescence

• Shining ultraviolet radiation on the specimen cause it to give off light that will form its own image, usually an intense red, blue, or green against a black field

• Has its most useful applications in diagnosing infections and pinpointing particular cellular structures

Transmission Electron Microscopy (TEM)

the method of choice for viewing the detailed structure of cells and viruses

• Produces its image by transmitting electrons through the specimen

Fresh, Living Preparations

Placed on wet mounts or in hanging drop mounts to observe as close to the natural state as possible.

- Cells are suspended in water, broth, or saline to maintain viability and provide a medium for locomotion

wet mount

consists of a drop or two of culture placed on a slide and overlaid with a cover slip

simple stain

only require a single dye and an uncomplicated procedure

- Cause all the cells in the smear to appear more or less the same color, regardless of type

• Reveal shape, size, and arrangement

Differential Stains

use two differently colored dyes: the primary dye and the counterstain

- Distinguish cell types or parts

• More complex and require additional chemical reagents to produce the desired reaction

Gram stain 1 (differential)

Developed in 1884 by Hans Christian Gram

Consists of sequential applications of:

• Crystal violet (the primary stain)

• Gram's iodine (the mordant)

• An alcohol rinse (decolorizer)

• A contrasting counterstain (for example safranin)

Gram stain 2

Different results in the Gram stain are due to differences in the structure of the cell wall (peptidoglycan and lipids)and how it reacts to the series of reagents applied to the cells

• Remains the universal basis for bacterial classification and identification

• A practical aid in diagnosing infection and guiding drug treatment

acid fast stain (differential)

Differentiates acid-fast bacteria (pink) from non-acid-fast bacteria (blue)

Originated as a method to detect Mycobacterium tuberculosis:

• These bacterial cell walls have a particularly impervious cell wall that holds fast (tightly or tenaciously) to the dye(carbol fuschin) when washed with an acid alcohol decolorizer

- Also used for other medically important bacteria, fungi, and protozoa