UNIT 6. CULTURE AND CULTURE MEDIA

CULTIVATION

Process of growing microorganisms in culture by taking bacteria from the infection site (in vivo environment) by some means of specimen collection and growing them in the artificial environment of the laboratory (in vitro environment)

CULTURE MEDIUM

Nutrient material prepared for the growth of microorganisms in a laboratory

• Carbon

• Nitrogen

• Sulfur

• Phosphorous

• Hydrogen

• Oxygen

• Buffer

ENUMERATE

composition of mixture of nutrients present in a culture medium

False

• Treponema pallidum

• Mycobacterium leprae _{^{(grows in mouse footpad)}}

• Chlamydia trachomatis

• Rickettsia

T or F

all bacteria can grown in an agar medium

inhibitory agents

this is used to make culture media selective

Haemophilus influenzae

which bacteria requires X and V factor for culture?

INOCULUM

Microbes that are introduced into a culture medium to initiate growth

CULTURE

Microbes that grow and multiply in or on a culture medium

1. Pure (Axenic) Culture

2. Mixed Culture

3. Stock Culture

ENUMERATE

types of CULTURE

Pure (Axenic) Culture

a culture that contains only one microbial species.

Mixed Culture

a culture that contains two or more microbial species.

Stock Culture

is a type of culture that are stored or maintained for future use in such a fashion that their growth and productive capabiliteis remains unalterediscapabilities remain

Stock Culture

Grown in a large volume of broth and then divided into small freezer vials (lengthen the shelf life of specimen to at least a year)

AGAR

Sulfated polymer made up of D-galactose, 3,6 anhydro-L-galactose, and D-glucoronic acid and usually derived from red algae

• D-galactose

• 3,6 anhydro-L-galactose

• D-glucoronic acid

AGAR is made up of?

80°C-90°C (100°C)

AGAR melts at what temp

40°C-50°C

AGAR solidifies at what temp

55°C-60°C

Cooling temperature for distribution of culture medium into Petri plates

20-25 ml

Amount of molten agar transferred to a sterile plates

1. Liquid medium

2. Semi-solid medium

3. Solid medium

ENUMERATE

classification of culture media

LIQUID MEDIUM

Does not contain any amount of agar or solidifying substances

• aerobes

• anaerobes

• facultative anaerobes

LIQUID MEDIUM allows growth of?

1. Nutrient Broth

2. Brain Heart Infusion (BHI)

3. Trypticase Soy Broth (TSB)

4. Thioglycollate (THIO)

[No Big TiTe]

ex of LIQUID MEDIUM

0.5% to 1% agar

agar content of SEMI-SOLID MEDIUM

SEMI-SOLID MEDIUM

used to observe bacterial motility and detect indole and sulfide production

Sulfide Indole Motility (SIM) Medium

[SIMi solid medium]

ex of SEMI-SOLID MEDIUM

2% to 3% agar

agar content of SOLID MEDIUM

Kovacs reagent

a biochemical solution used to test for indole production by microorganisms

red

color for indole producers

positive for tryptophanase

tryptophanase

a bacterial enzyme that catalyzes the hydrolysis of the amino acid tryptophan into indole, pyruvate, and ammonia

black precipitate

color for hydrogen sulfide production

1. Triple Sugar Iron (TSI) Agar

2. MacConkey (MAC) Agar

3. Blood Agar Plate (BAP)

4. Chocolate Agar Plate (CAP)

[TaMa Ba Co?]

ex of SOLID MEDIUM

tubes

solid medium should be placed on?

(TSI) Triple Sugar Iron Agar

a solid medium that is used for enterobacteriaceae detection, H₂S and gas production

1. SYNTHETIC OR DEFINED MEDIUM

2. NON-SYNTHETIC OR COMPLEX MEDIUM

3. TISSUE CULTURE MEDIUM

ENUMERATE

types of medium according to COMPOSITION

• cyanobacteria

• chemoorganotrophs

SYNTHETIC OR DEFINED medium is preferred for the isolation of what bacte?

SYNTHETIC OR DEFINED MEDIUM

Used for research purposes as either a liquid or a solid medium

SYNTHETIC OR DEFINED MEDIUM

Exact chemical composition of the ingredients is known (commercially prepared culture media)

BG-11 (Blue Algae) medium

ex of SYNTHETIC OR DEFINED MEDIUM

NON-SYNTHETIC OR COMPLEX MEDIUM

Precise composition of some or all the nutritive substances used is not known (Peptone, Meat and Yeasts Extracts)

medically significant bacteria

NON-SYNTHETIC OR COMPLEX MEDIUM medium is preferred for the isolation of what bacte?

1. Nutrient Broth (NB) Medium

2. TSB

3. MAC Agar

ex of NON-SYNTHETIC OR COMPLEX MEDIUM

TISSUE CULTURE MEDIUM

types of medium that contains loving tissues

obligate intracellular bacteria

• Rickettsia

• Chlamydia

TISSUE CULTURE MEDIUM is preferred for the isolation of what bacte?

• Mycobacterium leprae

• Rickettsia

• Treponema pallidum

• Chlamydia

[MoRe Tissue Culture]

examples of bacteria requiring living cells as medium

1. HeLa 229

2. McCoy and W 138

3. Embryonated Egg

ex of TISSUE CULTURE MEDIUM

Chlamydia (human cervical tissue)

HeLa 229 is used for?

Chlamydia (fibroblast)

McCoy and W 138 is used for?

propagation of Rickettsia

Embryonated Egg is used for?

1. plated media

2. tubed media

ENUMERATE

types of medium according to the dispensing of distribution method

1. slant

2. butt-slant

3. butt

types of TUBED MEDIA

Slant

type of tubed media that is used for biochem reaction

Butt

type of tubed media that is used for for motility

1. TSI — Butt-slant

2. SIM — Butt

3. Simmon’s Citrate Agar (SCA) — Slant

4. Lysine Iron Agar (LIA) — Butt-Slant

ex of TUBED MEDIA

SIMPLE MEDIA, SUPPORTIVE MEDIA OR GENERAL PURPOSE MEDIA

Routinely used in the laboratory and without additional supplements

SIMPLE MEDIA, SUPPORTIVE MEDIA OR GENERAL PURPOSE MEDIA

Support growth of most non-fastidious bacteria to grow at natural rates, without providing advantage to any particular bacteria

SIMPLE MEDIA, SUPPORTIVE MEDIA OR GENERAL PURPOSE MEDIA

Usually composed of meat and soybean extracts

1. Nutrient Agar

2. Nutrient Broth

3. TSB

ex of SIMPLE MEDIA, SUPPORTIVE MEDIA OR GENERAL PURPOSE MEDIA

ENRICHMENT MEDIA

enhance the growth of particular organisms (pathogens) and suppress the growth of normal flora present in specimen

ENRICHMENT MEDIA

incubated for a certain period and then subcultured to isolate the desired organism. contain specific nutrients and without additional supplements

ENRICHMENT MEDIA

(THIOGLYCOLLATE)

can also be used as a supplement to agar plates to detect aerobes, anaerobes and microaerophiles

1. Alkaline Peptone Water

2. Selenite F

3. Thioglycollate

4. Tetrathionate

5. Gram-negative broth

6. Lim Broth (Todd Hewitt with CNA)

[TTASGL]

Enumerate examples of ENRICHMENT MEDIA

Alkaline Peptone Water (APW)

Promote growth of Vibrio spp. Before inoculation into Thiosulfate-Citrate-Bile-Salts (TCBS) Agar.

pH 8.5

Alkaline Peptone Water (APW) pH i s adjusted to

Salmonella

Selenite F is used for isolation of __________ from feces, urine and water sample

Thioglycollate

general support enrichment medium that promotes the growth of almost all non fastidious bacteria

• Dextrose, Vitamin K1, and Hemin

• 0.075% agar

• Resazurin

• Thioglycolic Acid

[DART]

ENUMERATE

components of THIOGLYCOLLATE

Resazurin

what is the oxidation-reduction indicator in thioglycollate medium?

Thioglycolic Acid

whats is the reducing agent in thioglycollate medium?

Salmonella and Proteus

Tetrathionate: Selective enrichment broth for the isolation of ______________

Bile Salt and Thiosulfate

suppresses the growth of other coliform bacilli

Salmonella and Shigella

Gram-Negative Broth used for isolation of ______________________

Sodium citrate

Sodium deoxycholate

what composition of Gram-negative broth inhibits G(+) bacteria

Mannitol

primary carbon source in Gram-negative broth

Group B Streptococcus

• S. agalacticiae

Lim Broth (Todd Hewitt with CNA) is used to grow___________________

presence of black precipitate

how to detect H2S production?

Streptococcus pyogenes

ENUMERATE

Group A Streptococcus (GAS)

Streptococcus agalactiae

ENUMERATE

Group B Streptococcus (GAS)

ENRICHED MEDIA

media with additional supplements necessary for growth of fastidious organisms. solid type media

• Vitamins

• Peptone

• Blood

• Serum

• Yeast Extract

[VP sara BiSaYa]

ENUMERATE

supplements of ENRICHED MEDIA

1. Blood Agar Plate (BAP)

2. Chocolate Agar Plate

ex of ENRICHED MEDIA

haemolytic pattern of bacteria

BAP is used to differentiate what

• Sheep (most compatible)

• Horse (fastidious)

• Rabbit (fastidious)

choices of blood for BAP

5% defibrinated

BAP contains _____________ blood

Chocolate Agar Plate

medium that contain blood has been chemically-treated or heat treated (80°C) to lyse the RBC

• Neisseria gonorrhoeae

• Haemophilus spp.

CAP is used for isolation of fastidious microorganisms such as?

Nicotinamide Adenine Dinucleotide (NAD)

V factor contains?

Hemin

X factor contains?

Clostridium perfringens

a bacteria that has a double zone of hemolysis

• inner Beta hemolysis: tetha toxins

• outer Alpha hemolysis: alphatoxins

explain the double zone of hemolysis in C. perfringens

Gas gangrene

disease caused by C. perfringens

Gamma hemolysis

no hemolysis is termed as

Enterococci

Ex of non hemolytic bacteria

• Alpha hemolysis (partial/incomplete)

• Beta hemolysis (complete & clear)

• Gamma hemolysis (no hemolysis)

3 types of hemolytic pattern

S. pneumoniae

bacteria exhibiting alpha hemolysis

• Group A streptococci : S. pyogenes

• Group B streptococci : S. agalacticiae

bacteria exhibiting beta hemolysis

strep throat

disease caused by S. pyogenes

DIFFERENTIAL MEDIA

type of media that allows the visualization of metabolic differences between groups of bacteria

DIFFERENTIAL MEDIA

type of media that allows the grouping of microbes based on different characteristics demonstrated on the medium

• BAP

• EOSIN METHYLENE BLUE (EMB)

• HEKTOEN ENTERIC AGAR (HEA)

• MACCONKEY AGAR

[BEHMbang hits Diff]

Enumerate examples of DIFFERENTIAL MEDIA