2 Lab Tools of Microbiology

Lecture Notes

culture

any growth that appears in or on the medium after incubation

• standard incubators 37 C

inoculation

introduction of microbes into media for culture

medium

nutrient containing environment in which microbes can multiply

sterile (aseptic technique)

free of microbes; requirement for any instrument used for sampling and inoculation

broth, stab, slant

What are the different physical forms of media? Also known as liquid, semisolid, and solid/ reversible to liquid

general purpose media

Complex media that contains mixture of ingredients that support wide variety of microbial life, grows board spectrum of microbes

• Ex: Nutrient, Tryptic Soy Agar, Brain and Heart infusion

enriched media

Contains complex organic substances (blood, serum, hemoglobin, or special growth factors) that fastidious (complex) bacteria require for growth

• growth factors: specific vitamins or amino acids

streptococcus

Blood agar media is used for _______ species

Mannitol Salt Agar

This type of enriched media is used for bacteria that are salt tolerant known as halophiles are halo-tolerant bacteria

• Include Staphylococcus species & Micrococcus species

• Media that contains selective agent sodium chloride

Agar

A complex polysaccharide isolated from red alga Gelidium

• solid at room temperature, liquefies at 100C & once liquified doesn’t solidify until it reaches 42C

selective media

A type of media that contains ingredients that encourage a specific type of organism while inhibiting other types of organisms to grow

differential medium

distinguishes between different bacterial species based on their biochemical properties, often leading to visible changes in the medium or colony appearance

• allow multiple types of microorganisms but display visible differences between colonies

selective and differential

Mannitol Salt Agar is a ______ and ______ media:

• ________ because Staphylococcus grows in the presence of salt (and salt inhibits growth of other microbes)

• _______ because the bacteria will ferment mannitol & turn it into an acid causing a color change

Blood Agar

A differential but NOT selective media

• plate is red, bacteria growth is white/ transparent = bacteria able to lyse the red blood cells

chocolate agar

Differential and non-selective media made of overcooked blood

• bacteria that can grow here is enriched & can grow on that media

Mueller tellurite

A medium with selective agent potassium tellurite

• used for isolation of Corynebacterium diphtheriae

Enterococcus faecalis broth

Medium that contains sodium azide and tetrazolium

• used for isolation of fecal enterococci

phenylethanol agar

Media containing phenylethanol chloride

• used for isolation of staphylococci & streptococci

tomato juice agar

Media that contains tomato juice and acid

• used for isolation of lactobacilli from saliva

MacConkey agar

Selective and differential media containing bile & crystal violet that inhibits growth of gram positive and encourages growth of gram negative

• differential: contains glucose, if bacteria ferments it will change the color of the agar

Salmonella/Shigella agar (SS agar)

Media that contains bile, citrate, and brilliant green

• used for isolation of salmonella & shigella

Lowenstein-Jensen

Media that contains the selective agent Malachite green dye

• used for isolation & maintenance of Mycobacterium

Sabouraud’s agar

Media that contains selective agent with a pH of 5.6 (acid)

• used for isolation of fungi-inhibits growth of bacteria

Beta

In blood agar plate, ____ occurs when blood cell was lyse and cleared out

alpha

In blood agar plate, ___ occurs when blood cell is partially lyse

Gamma

In blood agar plate, ____ occurs when the bacteria couldn’t lyse the RBCs and only enjoyed the nutrients that the media provided and grew

Chrom agar

media that is nonselective and differential

• ingredients encourage all types of microorganisms and media differentiates organisms by pigments

pink, navy blue, orange, light blue, light pink, white

In Chrom Agar, the agar causes bacteria to turn different colors:

• E. coli = _____

• Klebsiella = _____ _____

• Proteus = _____

• Enterococcus = _____ _____

• S. Saprophyticus = _____ _____

• S. aureus = ______

hydrogen sulfide gas

The photo shoes that the inoculated organisms produce _____ _____ ___ producing black pigment at the bottom of the tube

Gas

____ is produced in this tube which disrupts the agar causing it to lift, bubble, or crack

mixed culture

a container that hold two or more identified, easily differentiated species of microorganisms

contaminated culture

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

inspection

Entails looking under the microscope to discern morphology & any type of arrangement

• Looking at growth pattern in a broth or solid

Identification

To name the genus and species which requires biochemical tests that can determine fundamental chemical characteristics:

• nutrient requirements

• products given off during growth

• presence of enzymes

• mechanisms for deriving energy

Stain

Dyes that impart colors to cells by becoming affixed to them through a chemical reaction

• provides contrast

• make inconspicuous features stand out

Cationic and anionic

What are two types of dyes used in microbial staining?

Cationic (basic) dyes

Dyes that has positive charge, attracted to acidic, negatively charged components of bacterial cell walls

• Positive staining where the cell is dyed

• Several subtypes include: simple stains, differential stains, and special stains

• some sort of cell distortion

Anionic (acidic) dyes

Dyes that have a negative charge, repelled by acidic negatively charged components on bacterial cell wall

• dyes include Nigrosin & India ink

• subtypes of stains include capsule & spore

simple stain

Stain that requires a positively charged dye used to determine size, shape, & arrangement of bacteria

• stains include crystal violet, safranin, & fungal red

• bacteria must be heat fixed

Differential stain

stain that divides microorganisms into groups based on their staining properties

• use two stains to clearly contrast cell types or cell parts

Gram stain

Use crystal violet, iodine, and safranin (counterstain)

• used to display gram positive and gram negative bacteria

• purple = gram positive

• red/ pink = gram negative

Acid fast stain

Used when a cell has a waxy membrane which are harder to penetrate w/t a water based stain

• waxy cells appear red (__ ___)

• counterstain will appear blue

Capsule stain

Staining a “white halo” bc these structures can’t be stained; use 2 stains

• one negative stain which will make the ____ appear white (stains background)

• positive stain that stains the bacteria cells

flagellar stain

Stain with an alcohol component which allows flagella to form a precipitate that thickens it

Endospore

a portion of DNA in bacteria covered by many membranes, hard to stain

• bacteria produce this when it feels threatened or environment is unfavorable

Vegetative cell

viable cells that are easier to stain & can still make a copy of themselves, if there is an endospore within a ______ ____ it is still viable

• once endospore leaves cell, it can no longer replicate

endospore stain

stain used to distinguish endospores from vegetative cells

• genera Bacillus & Clostridium

Malachite green, Congo Red

In an endospore stain, the endospore has a greenish color bc of the stain _____ ___ & vegetative cells have a red stain because of _____ ____ (counterstain)

Subterminal

What is the location of this endospore?

Central

What is the location of this endospore?

terminal

What is the location of this endospore?