Lecture 4A - Culture Media, Biosafety, and Aseptic Technique Flashcards
is a nutrient-rich liquid or gel designed to support the growth of a target species or group of species
culture medium
culture medium is a ____ designed to support the growth of a target species or group of species
nutrient-rich liquid or gel
What ingredients do culture media typically contain
- water
- energy source
- nitrogen source
- mineral source
- special growth factors
culture media: water
dissolve mix
culture media: energy source
usually a carbohydrate (glucose/dextrose)
culture media: nitrogen source
- amino acids
- protein sources (peptone)
culture media: mineral salts
- MgSO4
- NaCl
- KH2PO4
simplest amino acid
glycine
Major components of a medium
- Hydrogen, carbon, nitrogen
- inorganic nutrients
- trace elements
hydrogen
donors and acceptors
~1-15 g/L
carbon
source
~1-20 g/L
nitrogen
source
~0.2 - 2 g/L
inorganic nutrients
Sulfur, Phosphorus
~50 mg/L
trace elements
0.1-1 µg/L
Additives of a medium
- growth factors
- solidifying agent
- solvent
- buffers
example of growth factors
- amino acids
- purines
- pyrimidines
(~50 mg/L) - vitamins
(~0.1-1 mg/L)
example of solidifying agent
agar
(8-20 g/L)
percent of added standard agar
1.5-2%
percent of added high quality agar
1.5-1.7%
- ensure that pH is maintained
- very critical
buffers
Three 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
solid medium
percent of agar used in solid medium
1.5-2.0% or
1.5-2.0 g/100mL of medium
agar is a ___ __ __ prepared from seaweeds
golden-yellow granular powder
agar melts at
80°C
agar gels at
<40°C
- soft custard-like consistency
- prepared with agar at concentrations of 0.8% or less
- useful for the cultivation of microaerophilic bacteria or for the determination of bacterial motility
semisolid medium
semisolid medium is useful for what?
- cultivation of microaerophilic bacteria
- determination of bacterial motility
percent of agar used in semisolid medium
0.8% or less
- 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
liquid (broth) medium
various purposes of liquid (broth) medium
- propagation of large number of organisms
- fermentation studies
- various other tests
examples of various other tests which uses liquid (broth) medium
- sugar fermentation tests
- MR-VR broth
- organisms which can survive in both oxygenated as well as the deoxygenated environment
- most adaptable organisms that have the capability to switch between aerobic and anaerobic types of respiration
Facultative anaerobe
Two types of culture media based on composition/ingredients
- complex
- defined/synthetic
- with one or more unknown components
- with complex ingredients
complex
example of complex ingredients
- peptone
- extracts
- hydrolysates
example of rich medium
- potato dextrose agar/broth
- nutrient agar/broth
components of nutrient broth
- beef extract (3.0 g)
- peptone (5.0 g)
- distilled water (1000mL)
- proportion and identity of all components are known
- pure chemical substances
- may not be as rich as CM
defined/synthetic
example of defined/synthetic medium
Azotobacter enrichment medium
Types of culture media based on purpose
- general purpose media
- enrichment media
- selective media
- differential media
- transport media
- anaerobic media
- support growth of many in a microbial group
- do not contain any special additives
general purpose media
support growth of most cultivatable bacteria
- nutrient agar (NA)
- tryptic soy agar (TSA)
support growth of most cultivatable fungi
potato dextrose agar (PDA)
percent of microorganisms that have never been cultivated
99%
- provide nutrients and conditions that favor the growth of a particular microbe
- promotoe growth and dominance of a sensitive organism from a mixed culture
enrichment media
enriching medium to target microbial group with particular __ __
metabolic capacity
what makes an enriched medium
addition of extra nutrients to basal medium
what bacteria are grown in enriched media
fastidious bacteria
examples of enrichment media
- blood agar
- chocolate agar
- Loeffler’s serum slope
blood agar
adding 5-10% blood to blood agar base
chocolate agar
- heated blood agar
- lysed blood agar
first used enrichment to study chemolithotrophy
Sergei Winogradsky
- promote growth of organisms with the use of a favorable carbon source
- reduce the growth of other organisms with the use of inhibitory substances
selective media
added to medium to eliminate unwanted microbes
inhibitory substance
examples of inhibitory substances
- antibiotics
- dyes
- chemicals
- alteration of pH
Type of medium of selective media
agar-based (solid)
examples of selective media
- Sabouraud’s dextrose agar (SDA)
- Phenylethyl Alcohol Agar (PEA)
- Thayer Martin Medium
- Eosin Methylene Blue Agar (EMBA)
- Campylobacter Agar (CAMPY)
- Lowenstein-Jensen Medium
- high concentrations of sugar promote yeast growth and inhibit bacterial growth
- low pH also inhibits bacteria
Sabouraud’s dextrose agar (SDA)
- promotes growth of Gram positive cocci
- alcohol content tends to inhibit Gram-negative organisms
Phenylethyl Alcohol Agar (or Phenylethanol Agar) (PEA)
pH level of Sabouraud’s dextrose agar (SDA)
pH 6
- coumarin-derived antibiotic obtained from Streptomyces niveus
- antibacterial agent, an EC 5.99. 1.3 [DNA topoisomerase (ATP-hydrolysing)] inhibitor
- antimicrobial agent, an Escherichia coli metabolite and a hepatoprotective agent
Novobiocin
selective for Neisseria gonorrhoeae STD
Thayer Martin Medium
combination of antibiotics in Thayer Martin medium
- vancomycin
- colistin
- nystatin
- trimethoprim
Thayer Martin medium:
kill most Gram-positive organisms
vancomycin
Thayer Martin medium:
kill most Gram-negative organisms except Neisseria
colistin
Thayer Martin medium:
kill most fungi
Nystatin
Thayer Martin medium:
inhibits Gram-negative organisms, esp. Proteus
Trimethoprim
- selective for Gram-negative bacteria
- inhibit growth of Gram-positive bacteria
- also contain dye eosin Y
Eosin methylene blue agar (EMBA)
what inhibits growth of Gram-positive bacteria in eosin methylene blue agar (EMBA)
methylene blue
what dye does eosin methylene blue agar (EMBA) also contain
eosin Y
sugar in eosin methylene blue agar (EMBA)
sucrose
lactose
bacteria isolated in eosin methylene blue agar (EMBA)
enterobacteriaceae
- isolation of Campylobacter jejuni from fecal or rectal swab
- contain bacteriological charcoal, cefoperazone, and amphotericin B
Campylobacter Selective Agar (CAMPY)
what is isolated in Campylobacter Selective Agar (CAMPY)
Campylobacter jejuni
what does the Campylobacter Selective Agar (CAMPY) contain
- bacteriological charcoal
- Cefoperazone
- Amphotericin B
eosin methylene blue agar (EMBA):
what bacteria is possible to differentiate due to the presence of sugars lactose and sucrose
enteric bacteria
Bacteria isolated in eosin methylene blue agar (EMBA)
- lactose-fermenting gram-negative bacteria
- slow lactose fermenters
- non-lactose fermenters
lactose-fermenting gram-negative bacteria
green metallic sheen
slow lactose fermenters
brown-pink colonies
non-lactose fermenters
colorless or light pink colonies
- acidify lactose
- vigorous lactose and/or sucrose fermentation ability typical of fecal coliforms
- dark purple complex associated with green metallic sheen
lactose-fermenting gram-negative bacteria
less acidify lactose
slow lactose fermenters
- increase pH
- use sucrose in aerobic respiration
- colorless/light pink colonies
- eosin & methylene blue combine to form dark purple preciptate at low pH
non-lactos fermenters
components of Campylobacter Blood Agar (CVA)
- peptones extracts
- yeast extract
- dextrose
- sheep blood
- antimicrobial agents
- agar
Campylobacter Blood Agar (CVA):
supplies all the necessary growth factors
peptones extracts
Campylobacter Blood Agar (CVA):
source of B vitamins
yeast extracts
Campylobacter Blood Agar (CVA):
energy source
dextrose
Campylobacter Blood Agar (CVA):
hemin and supplies other necessary additional growth factors
sheep blood
Campylobacter Blood Agar (CVA):
- suppress the growth of the normal microbial flora in flecal speciments, therby facilitation isolation of C. jejuni
- cephalothin, polymycin B, trimethoprim, amphtericin B, and vancomycin
antimicrobial agents
what are the antimicrobial agents in Campylobacter blood agar (CVA)
- cephalothin
- polymycin B
- trimethoprim
- amphotericin B
- vancomycin
Campylobacter Blood Agar (CVA):
solidifiying agent
agar
- thermophiles
- inoculated plates should be incubated at 42C to accelerate growth
- higher temp. also inhibit background flora
Campylobacter jejuni
temp. for accelerated growth of Campylobacter jejuni
42 C
- solid medium used for Mycobacterium tuberculosis
- contain penicillin, nalidixic acid, and malachite green to inhibit growth of gram positive and gram negative bacteria, in order to limit growth to Mycobacteria species only
Lowenstein-Jensen Medium
inhibit growth of Gram positive and Gram negative bacteria in Lowenstein-Jensen Medium
- penicillin
- nalidixic acid
- malachite green
- allow identification of microorganisms usually through the (visible) phsiological reactions unique to those bacteria
- almost always selective
differential media
most practical media are those that both what
select for and differentiate common pathogens
differential media contain an __ that changes __ depending on the metabolites produced by the culture on the medium during incubation
- indicator
- color
Examples of differential media
- Desoxycholate Agar
- MacConkey Medium
- Xylose Lysine Deoxycholate Agar (XLD)
- Urease Medium
- Thiosulfate-citrate-bile salts-sucrose agar (TCBS)
- recognize the presence of Gram-negative enteric bacteria that ferment the milk sugar lactose
- these bacteria produce red pigment in the medium because of the acid it produce
Desoxycholate Agar
pigment produced by Gram-negative enteric organisms in Desoxycholate Agar
red pigment
- differentiate lactose fermenters from non-lactose fermenters
- only grows gram-negative bacterial species
- can further differentiate the gram-negative organisms based on their lactose metabolism
MacConkey Medium
components of MacConkey Medium
- Peptone
- Lactose
- Agar
- Neutral red
- Taurocholate
used for the recovery of Salmonella and Shigella species
Xylose Lysine Deoxycholate Agar (XLD)
Xylose Lysine Deoxycholate Agar (XLD):
- fermented by all enteric
- to enrich
xylose
Xylose Lysine Deoxycholate Agar (XLD):
- enable Salmonella to differentiate with nonpathogens
- attached by lysine decarboxylase
lysine
Xylose Lysine Deoxycholate Agar (XLD):
generates acid products when degraded causing color change to medium
xylose
lactose
sucrose
added to visualize hydrogen sulfide
- sodium thiosulfate
- ferric ammonium citrate
Xylose Lysine Deoxycholate Agar (XLD):
hydrogen sulfide
black (neutral/pH alkaline)
Xylose Lysine Deoxycholate Agar (XLD):
pH scale below 7
From phenol red to yellow
- tests the ability of an organism to produce an exoenzyme, called urease, that hydrolyzes urea to ammonia and carbon dioxide
- contains two pH buffers, urea, a very small amount of nutrients for the bacteria, and the pH indicator phenol red
Urease Medium
Urease Medium:
presence of urease (basic)
pink
- highly selective for the isolation of Vibrio cholerae
and V. parahaemolyticus - very alkaline (8.5-9.5)
Thiosulfate-citrate-bile salts-sucrose agar (TCBS)
carbon source of Thiosulfate-citrate-bile salts-sucrose agar (TCBS)
sucrose
dye differentiation in Thiosulfate-citrate-bile salts-sucrose agar (TCBS)
- bromothymol blue
- thymol blue
what does light blue indicate in Thiosulfate-citrate-bile salts-sucrose agar (TCBS)
V. parahaemolyticus
pH level of Thiosulfate-citrate-bile salts-sucrose agar (TCBS)
8.5 - 9.5
- media used for transporting samples
- non nutritional (non growing)
- isotonic
transport media
transport media can maintain __ of micoorganisms without __
- viability
- multiplication
what does transport media prevent
oxidizing and drying
components of transport media
- sodium thioglycolate
- sodium glycerophosphate
- saline (calcium chloride)
- methylene blue
Transport media:
sequester oxygen
sodium thioglycolate
Transport media:
oxygen diffusor
colorless (if without oxygen)
methylene blue
Transport media:
permeability
saline (calcium chloride)
Transport media:
maintain pH
sodium glycerophosphate
used to grow anaerobic organisms
anaerobic media
Examples of anaerobic media
- Robertson’s cooked meat medium
- Thioglycolate broth medium
- nutrient broth with meat pieces
- meat pieces create small packets of anaerobic habitats
Robertson’s cooked meat medium
- used for testing oxygen requirement of bacteria
- contains resazurin (will turn pink due to presence of oxygen)
Thioglycolate broth medium
medium will turn pink due to presence of oxygen
indicator resazurin
Concept Check:
culture media can be manipulated to target the growth and isolation of specific microorganisms
true
Concept Check:
enrichment media can be used to allow particular growth of microbes to dominate culture
true
Concept Check:
indicators are used to differentiate species growin on differential media
true
Concept Check:
PDA - bacteria
NA - fungi
false
Concept Check:
all microbes are cultivatable
false