Lecture 4A: Culture Media, Biosafety and Aseptic Technique Flashcards
Culture _____ (sing.)
medium
Culture ____ (plu.)
media
_____ - is a nurtient-rich liquid or gel designed to support the growth of a target species or group of species.
Culture medium
is a ______________________ designed to support the growth of a target species or group of species.
nutrient-rich liquid or gel
What does culture medium/media typically contain?
- water
- energy source, usually a carbohydrate
- nitrogen source (eg. amino acids, protein sequences such as pepton)
- mineral salts (MgSO4, NaCl, KH2PO4)
- special growth factors (if needed)
General composition of medium;
- H-donors and acceptors
- C-source
- N-source
- Inorganic nutrients
- Trace elements
- Growth factors
- Solidifying agent
- Solvent (usually distilled water)
- Buffers
General composition of medium;
H-donors and acceptors
~1-15 g/L
General composition of medium;
C-source
~1-20 g/L
General composition of medium;
N-source
~0.2-2 g/L
General composition of medium;
Inorganic nutrients
e.g. S, P, (~50mg/L)
General composition of medium;
Trace elements
(0.1-1 μg/L
General composition of medium;
Growth factors
- aminoacids, purines, pyrimidines, occasionally 50 mg/L,
- vitamins occasionally 0.1-1 mg/L
General composition of medium;
Solidifying agent
- eg. Agar (8-20 g/L) (if semi-solid or solid medium)
(3)Types of culture media based on physical state
- Solid medium
- semisolid medium
- liquid (broth) medium
- for isolation of colonies , which give distinct morphology on the surface of the medium
- commonly employs agar as the solidifying agent, added at 1.5-2% (or 1.5-2.0 g/100ml of medium)
- agar is a golden-yellow granular powder prepared from seaweeds
Solid medium
Solid medium melts at ___ and gels at ____
melts at 80°C and gels at <40°C
- prepared with agar at concentrations of
0.8% or less. - has a soft custard-like consistency and is useful for the cultivation of microaerophilic bacteria or for the determination of bacterial motility.
Semisolid medium
Semisolid medium has a soft custard-like consistency and is useful for the cultivation of _______ _______ or for the ____ _ ____ ____.
-microaerophilic bacteria
- determination of bacterial motility
- contain specific amounts of nutrients but do not contain solidifying agent.
- serves various purposes such as propagation of a large number of organisms, fermentation studies, and various other tests. e.g. sugar fermentation tests, MR-VR broth
liquid (broth) medium
- serves various purposes such as propagation of a large number of organisms, fermentation studies, and various other tests. e.g. __ ____ __, __ __
- sugar fermentation tests, MR-VR broth
Microoganism can be classified depending on ____
oxygen requirements
Classifications of microorganisms
- Aerobic
- Facultative
- Anaerobic
- Capnophilic
- Microaerophilic
requires O2
- aerobic
can grow in the presence or absence of O2
- facultative
growth in the absence of O2
- anaerobic
CO2 stimulates growth
capnophilic
require slightly decreased O2 tenstion
microaerophilic
(2) Types of culture media based on composition / ingredients
- Complex
- Defined /synthetic
with 1 or more unknown components, with complex ingredients
- eg. peptone, extracts, hydrolysates; rich medium
Complex
examples of complex media
- potato dextrose agar/broth
- nutrient agar/broth
proportion and identity of all components are known; pure chemical substances; may not be as rich as CM
Defined / Synthetic
Types of culture media based on purpose
- General Purpose media
- Enrichment media
- Selective media
- Differential Media
- Transport media
- Anaerobic media
- support the growth of many in a microbial group
- They do not contain any special additives.
General purpose media
General Purpose media- ____ ___ ___ any special additives
do; not; contain
support growth of most cultivatable bacteria
- Nutrient agar (NA)
- Tryptic soy agar (TSA)
Nutrient agar (NA) and tryptic soy agar (TSA) support growth of most cultivatable _____________
bacteria
supports growth of most cultivatable fungi
Potato dextrose agar (PDA)
Potato dextrose agar (PDA) supports growth of most cultivatable _________.
fungi
-provide nutrients and conditions that favor the growth of a particular microbe.
- promote growth and dominance of a sensitive organism from a mixed culture.
Enrichment Media
to target microbial group with particular metabolic capacity.
Enriching medium
Enriching medium to target microbial group with particular ____________ ________________.
-metabolic capacity
Enriched media are used to grow nutritionally exacting (____________) bacteria.
fastidious
example of enriched medium
- chocolate agar
- blood agar
- Loeffler’s serum slope
also known as heated blood agar or lysed blood agar
chocolate agar
Chocolate agar or ______
Blood agar
from sheep (usually) created by adding 5-10% (by volume) blood to blood agar base
Blood agar
first used enrichment to study Chemolithotrophy
- Sergei Winogradsky
- promote the growth of organisms with the use of a favorable carbon
source - and will reduce the growth of other organisms with the use of inhibitory substances
Selective media
The ________________ is added to medium to eliminate unwanted microbes
inhibitory substances
example of inhibitory substances
- Antibiotics
- Dyes
- Chemicals
- Alteration of pH
Principle of selective media
Differential growth suspension
state the 2 The principle of selective media’s Differential growth suspension
- is designed to suppress growth of some microorganisms, while allowing the growth of others
- is agar-based (solid) medium so that individual colonies may be isolated
Examples of Selective media
- Sabouraund’s Dextrose agar (SAB)
- Phenylethyl Alcohol Agar (Phenylethanol agar)
- High concentrations of sugar
promote yeast growth and inhibit bacterial growth. - In addition, low
pH also inhibits bacteria
Sabouraund’s Dextrose agar (SAB)
- Promotes the growth of Gram positive cocci
- Promotes the growth of Gram positive cocci
Phenylethyl Alcohol Agar (Phenylethanol agar)
active against fungi, fungi cant grow it a medium with novobiocin
Novobiocin
- selective for Neisseria gonorrhoeae
Thayer Martin Medium
Combination of antibiotics in Thayer Martin medium
- Vancomycin
- Colistin
- Nystatin
- Trimethoprim
(antibiotic) which is able to kill most Gram-positive organisms
Vancomycin
which is added to kill most Gram-negative organisms EXCEPT Neisseria.
Colistin
(antibiotic) which can kill most FUNGI
Nystatin
which inhibit gram-negative organisms, especially swarming ‘PROTEUS’
Trimethoprim
selective for gram negative bacteria
Eosin methylene blue
is used for isolation of Camplyobacter jejuni from fecal or rectal swab
Camplybacter agar
Camplybacter agar is used for isolation of _____ ____ from fecal or rectal swab
Camplyobacter jejuni (scientific name)
contains sucrose and lactose, utilized as fermentable carbohydrates substrates, which encourage the growth of some gram-negative bacteria, especially fecal and non-fecal coliforms.
EMB agar
EMB agar contains __ and __, utilized as fermentable carbohydrates substrates, which encourage the growth of some gram-negative bacteria, especially fecal and non-fecal coliforms.
sucrose and lactose
is possible due to the presence of the sugars lactose and sucrose and the ability of certain bacteria to _____________ the
lactose in the medium.
Differentiation of enteric bacteria
green metallic sheen.
Lactose-fermenting gram-negative bacteria
brown-pink colonies
- produces less acid.
Slow lactose-fermenters
colorless or light pink colonies
- increase the pH of the medium by deamination of proteins.
Non-lactose fermenters
Campylobacter Blood Agar ; supplies all the necessary growth factors
Peptone and extracts
Campylobacter Blood Agar ; source of B vitamins
yeast extracts
Campylobacter Blood Agar ; energy source
dextrose
Campylobacter Blood Agar ; hemin and supplies other necessary
additional growth factors.
Sheep blood
Campylobacter Blood Agar ; cephalothin, polymyxin B, trimethoprim, amphotericin B, and vancomycin, suppress the growth of the normal microbial flora in fecal specimens, thereby facilitating isolation of C. jejuni.
Antimicrobial agents
Solidfying agent
Agar
Solid medium used for Mycobacterium tubercolosis
- contain penicillin, nalidixic acid and malachite grene to inhibit growth of gram-positive and gram-negatuve bacteria, in order to limit growth to mycbacteria species only.
Lowenstein-Jenson medium
Lowenstein-Jenson medium contains __, __ __ and __ __.
- Pencillin
- Nalidix acid
- malachite green
allow identification of microorganisms usually through the (visible) physiological reactions unique to those bacteria.
Differential Media
The most practical media are those that both select for and differentiate common pathogens Usually contain an __________ that changes __________ depending on the metabolites produced by the culture on the medium during incubation
- Indicator
- color