Lecture 4A - Culture Media, Biosafety, and Aseptic Technique Flashcards by Ahn Studies

is a nutrient-rich liquid or gel designed to support the growth of a target species or group of species

culture medium

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culture medium is a ____ designed to support the growth of a target species or group of species

nutrient-rich liquid or gel

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What ingredients do culture media typically contain

water
energy source
nitrogen source
mineral source
special growth factors

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culture media: water

dissolve mix

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culture media: energy source

usually a carbohydrate (glucose/dextrose)

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culture media: nitrogen source

amino acids
protein sources (peptone)

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culture media: mineral salts

MgSO4
NaCl
KH2PO4

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simplest amino acid

glycine

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Major components of a medium

Hydrogen, carbon, nitrogen
inorganic nutrients
trace elements

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hydrogen

donors and acceptors
~1-15 g/L

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carbon

source
~1-20 g/L

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nitrogen

source
~0.2 - 2 g/L

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inorganic nutrients

Sulfur, Phosphorus
~50 mg/L

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trace elements

0.1-1 µg/L

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Additives of a medium

growth factors
solidifying agent
solvent
buffers

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example of growth factors

amino acids
purines
pyrimidines
(~50 mg/L)
vitamins
(~0.1-1 mg/L)

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example of solidifying agent

agar
(8-20 g/L)

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percent of added standard agar

1.5-2%

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percent of added high quality agar

1.5-1.7%

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ensure that pH is maintained
very critical

buffers

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Three types of culture media based on physical state

solid medium
semisolid medium
liquid (broth) medium

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for isolation of colonies which give distinct morphology on the surface of the medium

solid medium

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percent of agar used in solid medium

1.5-2.0% or
1.5-2.0 g/100mL of medium

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agar is a ___ __ __ prepared from seaweeds

golden-yellow granular powder

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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

1. complex 2. 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

1. general purpose media 2. enrichment media 3. selective media 4. differential media 5. transport media 6. 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

1. antibiotics 2. dyes 3. chemicals 4. alteration of pH

Type of medium of selective media

agar-based (solid)

examples of selective media

1. Sabouraud's dextrose agar (SDA) 2. Phenylethyl Alcohol Agar (PEA) 3. Thayer Martin Medium 4. Eosin Methylene Blue Agar (EMBA) 5. Campylobacter Agar (CAMPY) 6. 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

1. vancomycin 2. colistin 3. nystatin 4. 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)

1. lactose-fermenting gram-negative bacteria 2. slow lactose fermenters 3. 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)

1. peptones extracts 2. yeast extract 3. dextrose 4. sheep blood 5. antimicrobial agents 6. 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)

1. cephalothin 2. polymycin B 3. trimethoprim 4. amphotericin B 5. 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

1. penicillin 2. nalidixic acid 3. 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

1. Desoxycholate Agar 2. MacConkey Medium 3. Xylose Lysine Deoxycholate Agar (XLD) 4. Urease Medium 5. 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

1. Peptone 2. Lactose 3. Agar 4. Neutral red 5. 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

1. sodium thioglycolate 2. sodium glycerophosphate 3. saline (calcium chloride) 4. 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

1. Robertson's cooked meat medium 2. 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