Tools of the Laboratory: media

why are bacterial cultures important

for accurate diagnostics

what do accurate diagnostics rely on

appropriately grown microorganisms

what do bacterial cultures must have

a pure culture

what is present in a pure culture 

Only a single bacterial specie

What is culture considered

clonal

How do microbiologists ensure a pure culture

• Starts with a good sample

• Selection of the appropriate growth medium

• Proper culture technique

Name the three general categories of media.

• physical state (medium normal consistency )

• Chemical composition (type of chemical medium contains)

• functional type (purpose of medium)

liquid media 

bacteria is suspended throughout media 

what does liquid media support

growth to high density (can grow huge amounts of bacteria)

what media is hard to obtain pure cultures

liquid media

what media is used to determine motility

semisolid

solid media

firm surface on which cell can form discrete colonies

what is solid media advantage

advantageous for microbial infection

what type of media has slow bacterial growth and limited to surface area

solid media

What determines the physical state of media

solidfying agents, useful properties

solidifying agents 

• Solid > Semisolid > Liquid

• Lots > Some > None

useful properties of media

• Liquefies at high temperatures (will conform to the shape of the container)

• Solid at culturing temperature

• Not digestible for most microorganisms

most common media

agar

agar

Polysaccharide derived from red seaweed

Synthetic or Chemically Defined media

Exact formula with defined molecular content

• Standardized - very little variation from batch-to-batch

for each ingredient need a chemical formula 

What are chemically defined media useful for

research and for niche tests

Synthetic or Chemically Defined media cons

• Difficult to make

• Must know the nutritional requirements of organism

Nonsynthetic or Complex

if any component of the media is not described by an exact chemical formula 

• Composition varies from batch-to-batch

Nonsynthetic or Complex pros

• Provide rich mixture of nutrients for microbes with complex needs.

• Supports many types of organisms

selective media 

Contains component(s) that inhibit the growth of certain microbes but

not all microbes

what is selective media important for

isolation of microorganisms

differential media

Designed to show visible differences between different groups of organisms

what could differential media manifest as depending on the medium

colony size/color changes, media color changes, or formation of gas or precipitates

Why is sterilizing media necessary

to avoid unwanted growth

two methods of sterilizing media

autoclave and filter

autoclave

High temperature and pressure

• common lab equipment

what are autoclaves useful for

sterilizing large volumes

media components in an autoclave cannot be

heat labile

filter

Media suctioned through very small pores that bacteria are unable to pass through

what is the most common size of components goes through the media

22um

filter con

Expensive and time consuming for large volumes

inoculation

Introduction of a small sample of microorganisms (the inoculum) into a container of nutrient medium.

culture is the result of

The organisms multiplying

isolation

If an individual cell is separated from other cells

when will a colony grow

when an individual cell is separated and provided adequate space, it will grow into a discrete group of cells (colony)

• streak plate

• dilutions

how can a colony form

Small number of cells must be inoculated onto a relatively large, solid surface

Specific media

• Luria Bertani broth and agar

• Blood agar plates (BAP)

• Eosin-methylene blue (EMB) agar

Blood agar plates (BAP)

General-purpose, enriched media used to grow fastidious organisms

what is blood agar plates

a differential media

what does blood agar plates allow for

the identification of bacteria based on their ability to hemolyze red

blood cells

Eosin-methylene blue (EMB) agar

Selective and differential media

what type of bacteria does Eosin-methylene blue (EMB) agar isolate

gram negative bacteria

Eosin-methylene blue (EMB) agar: methylene blue

inhibits growth of gram positve bacteria

Eosin-methylene blue (EMB) agar: eosin

dye that reacts with organic acids secreted by growing bacteria resulting in a color change to dark purple.

pure culture

Growth of a single species of microbe

contamination

Presence of microbes other than those in the sample

when does contaimination occur

during collection, transport, and culture

bacteria growth conditions

physical requirements

chemical requirements 

physical requirements

temp

pH

osmotic pressure

chemical requirements

CHONPS

trace elements

oxygen

organic growth factors

regarding temp each organism has a

• Maximum growth temperature

• Minimum growth temperature

• Optimum growth temperature

Psychrophiles

cold loving 

Mesophiles

moderate temperature

Thermophiles

heat loving

example of extreme thermophiles

archaea

80 degress C and above 

acidophiles

acid loving

neutrophiles

neutral loving

alkalophiles

base loving

Cellular metabolism produces

acid

what do acids do 

inhibit bacterial growth 

why are medias buffered

to stabilize pH

What is an innate host defense against invading microorganisms

bodily pH (stomach, skin)

what do microbes require 

water 

osmosis

Diffusion of water across a selectively permeable membrane

osmotic pressure categories

Isotonic : water in and outside equal

Hypotonic: alot of water inside the cell→ cell swells 

Hypertonic: little water in the cell→cell shrinks/ shivel 

• Food preservative

Halotolerant or facultative halophiles

• do not require salt for growth but can survive and thrive in salty environments 

Extreme halophiles or obligate halophiles

grow and thrive high salt concentration/ environment 

CHONPS

Carbon

hydrogen 

oxygen 

nitrogen

phophorous 

sulfer 

Carbon

backbone of all living matter and organic life

hydrogen and oxygen

found in all components of cells

Nitrogen

14% dry weight of cell (proteins and nucleic acids

phosphorous and sulfur

4% dry weight of cell (cysteine and nucleic acids)

trace elements

Small amounts of minerals

what are trace elements important as

cofactors for enxymes

examples of trace elements

iron, copper, zinc

oxygen is

essential as a final electron acceptor

what does oxygen result in

toxic oxygen radicals (free radicals)

• hydrogen peroxide 

what do macrophages produce

oxygen radicals as an antimicrobial defense mechanism

what does bacteria produce

detoxifying enzymes to combat radicals

lag phase

little or no cell division

• period of synthesis

log phase 

exponential growth 

• cells are most metabollically active in this stage 

what bacterial growth stage is most sensitive to antibiotics

log phase

stationary phase

• Nutrients are all utilized, pH change

• Constant number of cells

• Division rate = death rate

death phase

Death rate > division rate

No nutrients, extreme pH

bacterial quantification

Viable plate counts (similar to isolation)

Filtration

Most probable number

Microscopy

Turbidity

 Metabolic activity

Dry weight

Viable Plate Counts

counts live cells

what do Viable Plate Counts usally require

serial dilutions

what isnt Viable Plate Counts useful for

non-culturable organisms

Effects of dilutions

multiplicative

what is filtration used for 

small number of cells 

how to do filtration

Concentrate bacteria with a filter, then plate them to coun

when is filtration usally used 

to quantify coliforms (enteric bacteria) from sewage

• enteric bacteria examples: Salmonella and Escherichia coli

what does Microscopy do

directly count number of cells

what is Microscopy useful for

non-culturable cells

problem with Microscopy

• Motile cells

• Dead cells

• Time consuming