MCB 2004 Chapter 3 Tools of Lab Hartmann

The 5 I's

Inoculation, Incubation, isolation, inspection, identification

Central Dogma of Biology

DNA is transcribed into RNA which is translated into proteins

Our body temp in Celsius is

37 degrees C

polymicrobial infection

many microbes present at once, Ex: diabetic foot ulcer

Inoculation

Introduction of microbes into or on a medium (Ex: agar)

Incubation

Usually 20-45 degrees C / our cells like a 5% CO2

Listeria monocytogenes

causes listeriosis which is an intracellular parasite usually behind food poisoning, incubates from 25-30 degrees C

Streptococcus pyogenes

Strep throat, linked to OCD in kids

Mycobacterium tuberculosis

causes TB, can be prevented by intake of STEROID D, most of the lung tissue is actually killed by the immune response

Mycobacterium leprae

slowest reproduction of any known bacterium

Media: Providing Nutrients in Lab

1. Must be provided with all of their required nutrients in an artificial medium

2. Certain microbes cannot be grown in a medium and require cell cultures or host animals, known as non culturable bacteria, they are the majority

3. Use gene sequencing to find and identify

Different Types of Artificial Media examples

Brain-heart infusion is general purpose and enriched; mannitol salt agar and blood agar are both selective and differential

Selective Media

suppress growth of unwanted bacteria and encourage growth of desired microbes

Differential Media

allows growth of several types of microbes and displays visible differences among those microbes

T/F: Agar is a Medium

False: Agar is a solidifying agent not the media itself

Different kinds of Media: Liquid Media

don't solidify at temp above freezing and flow freely

Diff Kinds of Media: Semisolid

clot like consistency; contains enough gelatin or agar to thicken but not produce a firm surface; used to determine motility

Diff Kinds of Media: Solid

provides firm surface for cells to form a discrete colony and be able to isolate certain groups

Agar

a complex polysaccharide derived from a red alga and used as a solidifying agent in culture media, solid at room temp, liquifies at 100C and doesn't solidify until it goes under 42C

Defined Media

media who's composition is exactly known

Complex media

-Contains at least one component that is not chemically definable

-contains extracts of animals, plants, or yeast

-Ex: blood, serum, meat extracts, milk, yeast extract, soybean digest, peptone

• nutrient broth, blood agar, and MacConkey agar

General purpose media

-grows many types of microbes

-nutrient agar and broth, brain-heart infusion, andtrypticase soy agar

enriched media

-Contains complex organic substances (blood, serum,hemoglobin, or special growth factors) that fastidiousbacteria require for growth• -Growth factors: specific vitamins or amino acids

Selective and/or Differential Media

-extensive applications for isolation and identification

-Permit preliminary identification of a genus or even aspecies of bacteria

MacConkey Agar

-isolation of gram-negative enterics(microbes that live in the gut)

• selective agent is bile

Lowenstein-Jensen

isolation of Mycobacterium (rod shaped and gram positive)

mannitol salt agar

-isolation of Staphphylococcus species

• selective agent is salt

Sabourand's agar

-isolation of fungi

-inhibits bacteria

blood agar

-Used as an enrichment medium for fastidious microbes as well as differential media

fastidious microbe

Microorganisms that have complex nutritional requirements and will not grow readily on basal media without nutrient supplements

hemolysins

lyse red blood cells to release iron rich hemoglobin

beta hemolysis

-complete lysis of red blood cells

-example is strep pyogenes

alpha hemolysis

incomplete lysis of red blood cells

gamma hemolysis

no hemolysis

T/F: MacConkey Agar and Mannitol Salt Agar can both be selective and differential

True, MacConkey and Mannitol Salt are both selective and differential

Reducing media

grow anaerobic bacteria

Carbohydrate fermentation media

contain sugars that can be fermented and a pH indicator; useful for identification of microorganisms

assay media

used to test the effectiveness of antimicrobial drugs

enumeration media

used by industrial and environmental microbiologists to count the numbers of organisms in milk, water, food, soil, and other samples

isolation technique

goal is to obtain a pure colony, just one species

streak plate method

a method of isolating a culture by spreading microorganisms over the surface of a solid culture medium in quadrants

pour plate method

sample is diluted serially into cooled but still liquid agar tubes; inoculated tubes are poured into sterile Petri dishes & harden; diluted cells have enough space to grow in separate colonies

spread plate technique

small amount of sample is spread across media by "hockey stick" to form individual colonies

pure culture

contains only one species or strain

subculture

a second level culture from a well isolated colony

mixed culture

A container growing two or more different, known species of microbes.

contaminated culture

A culture that was once pure or mixed that now contains contaminants, or unwanted microbes of uncertain identity

biochemical tests

• determine products given off during growth

• determine presence of enzymes

genotypic testing

detects microbes based on their DNA

immunologic testing

testing the isolate against known antibodies

microbe sizes

• prions: around 10nm

• HIV: around 100nm

• Mycoplasma: 200nm

• E. coli: 1um/ micrometer(5x the size of mycoplasma)

• red blood cell: 10um/ micrometers

oil immersion

used to increase the resolving power of a microscope; lower lightwave wavelength, increasing numerical aperture

flourescence microscopy

uses UV light

confocal microscopy

uses a laser beam

Smear technique

• Spread a thin film made from a liquid suspension of cells on a slide

• Allow the slide to air dry

• Heat fixing

• Heat the slide gently after it has been air dried

Heat fixing

Heat the slide gently after it has been air dried

stained smears

coloring microorganisms with a dye that emphasizes certain structures

basic stain smear

-cationic

-have a positive charge, attracted to acidic and negatively charged components in cell wall

acidic stain smear

-anionic

-repelled by acidic, negatively charged components on bacterial cell wall

positive stain

dye sticks to the specimen and gives it color

negative stain

colors the background, which makes capsules more visible

positive stain characteristics

-dye used: crystal violet

-subtype: gram stain and acid-fast stain

negative stain characteristics

dye used: india ink, nigrosin

simple stain

requires only one dye

differential stain

-procedure that uses different colored stains to contrast different cell types or parts

• acid-fast and gram stain are examples

gram stain

-universal technique for staining bacteria

-gram positive is purple

-gram negative is pink

acid-fast stain

-bacteria stain pink

-detects agents of leprosy and tuberculosis

endospore stain

detects endospores of Bacillus and Clostridium

capsule stain

-Visualized through negative staining with India ink or special positive stains

-detects Cryptococcus

flagellar staining

-flagella must be enlarged to be seen