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

CULTURE MEDIUM IS COMPOSED OF

mixture of nutrients: Carbon, Nitrogen, Sulfur, Phosphorous, Hydrogen, Oxygen, and Buffer

Inhibitory agents

Facilitate isolation of desired organism while suppressing the growth of other organism

Used to make the culture media selective

INOCULUM

Microbes that are introduced into a culture medium to initiate growth

CULTURE

Microbes that grow and multiply in or on a culture medium

Pure (Axenic) Culture

Composed of only one species

Mixed Culture

Composed of more than one species

Stock Culture

Composed of several species contained in a separate culture medium (one specie per culture medium)

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

Melt at 80°C-90°C (100°C) and solidify at 40°C-50°C

Agar melts at ____ and solidifies at ____

D-galactose, 3,6- anhydro-L-galactose
D-glucoronic acid
red algae

Agar is sulfated polymer made up of _____, and ____ and usually derived from ____

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

LIQUID MEDIUM

Does not contain any amount of agar or solidifying substances
Allows growth of aerobes, anaerobes and facultative anaerobes

1. Nutrient Broth
2. Brain Heart Infusion (BHI)
3. Trypticase Soy Broth (TSB)
4. Thioglycollate (THIO

Term of Liquid Medium

SEMI-SOLID MEDIUM

Observed bacterial motility and detect indole and sulfide production

Contains 0.5% to 1% agar

Semi-solid medium contains how much agar

Sulfide Indole Motility (SIM) Medium

Examples of Semi-solid medium

2% to 3%

SOLID MEDIUM contains how much agar?

1. Triple Sugar Iron (TSI) Agar
2. MacConkey (MAC) Agar
3. Blood Agar Plate (BAP)
4. Chocolate Agar Plate (CAP)

examples of solid medium

SYNTHETIC OR DEFINED MEDIUM

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

Cyanobacteria and Chemoorganotrophs

SYNTHETIC OR DEFINED MEDIUM is preferred for the isolation of

NON-SYNTHETIC OR COMPLEX MEDIUM

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

1. Nutrient Broth (NB) Medium
2. TSB
3. MAC Agar

Examples of Non-synthetic medium

TISSUE CULTURE MEDIUM

Contains living tissues
Used for obligate intracellular bacteria – Rickettsia and Chlamydia

1. HeLa 229
2. McCoy and W 138
3. Embryonated Egg

TISSUE CULTURE MEDIUM examples

1. HeLa 229 – human cervical tissue, Chlamydia
2. McCoy and W 138 – fibroblasts, Chlamydia
3. Embryonated Egg – propagation of Rickettsia

1. HeLa 229 – _________
2. McCoy and W 138 – ______
3. Embryonated Egg – propagation of ________

PLATED MEDIA

Distributed into sterile petri dish

TUBED MEDIA

Distributed in sterile test tube

1. TSI
2. SIM
3. Simmon’s Citrate Agar (SCA)
4. Lysine Iron Agar (LIA)

Examples of Tubed media

SIMPLE MEDIA, SUPPORTIVE MEDIA OR GENERAL PURPOSE MEDIA

Routinely used in the laboratory and without additional supplements

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

Usually composed of meat and soybean extracts

1. Nutrient Agar
2. Nutrient Broth
3. TSB

examples of simple media

ENRICHMENT MEDIA

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

enrichment media

contain specific nutrients and without additional supplements

incubated for a certain period and then subcultured to isolate the desired organism

1. Alkaline Peptone Water (APW)
2. Selenite F
3. Thioglycollate
4. Tetrathionate
5. Gram-Negative Broth
6. Lim Broth (Todd Hewitt with CNA)

examples of enrichment media (6)

Alkaline Peptone Water (APW)

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

Adjusted to pH 8.5

Selenite F

Isolation of Salmonella from feces, urine and water sample

Thioglycollate

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

Resazurin
Thioglycolic Acid

____ → oxidation-reduction indicator

____ → reducing agent

Tetrathionate

Selective enrichment broth for the isolation of Salmonella and Proteus

Bile Salt and Thiosulfate

suppresses the growth of other coliform bacilli

Gram-Negative Broth

isolation of Salmonella and Shigella
Enrichment and Selective medium

Lim Broth (Todd Hewitt with CNA)

enrichment media for Group B Streptococc

Sodium Citrate and Sodium Desoxycholate
Mannitol

_____ (a bile salt) → inhibit gram-positive organism
_____ → primary carbon source

ENRICHED MEDIA

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

Blood, Vitamins, Serum, Peptone and Yeast Extract

Supplements of enriched media

Blood Agar Plate (BAP)

Contains 5% defibrinated blood
Differentiate haemolytic pattern of bacteria

Sheep, horse, rabbit
5%

choices of blood and amount of defibrinated blood of BAP

Chocolate Agar Plate

blood has been chemically-treated or heattreated (80°C) to lyse the RBC

Neisseria gonorrhoeae and Haemophilus spp.

chocolate agar plate is for the isolation of fastidious microorganisms like

DIFFERENTIAL MEDIA

allow the visualization of metabolic differences between groups of bacteria
distinguishes organisms growing together by their differences in cultural characteristics
allow grouping of microbes based on different characteristics demonstrated on the medium

BAP
EMB
HEA
MAC

examples of differential media

MacConkey Agar

differentiate Lactose Fermenter (pink colonies) from Non-Lactose Fermenter (colorless colonies)

1. Lactose
2. Bile Salts
3. Crystal Violet – inhibit gram-positive bacteria and fungi
4. Neutral Red – pH indicator

Components of MacConkey Agar

SELECTIVE MEDIA

support the growth of one type or group of microbes but not another

SELECTIVE MEDIA

contain inhibitory substances such as antimicrobials, dyes, or alcohol which inhibit the growth of other organisms while promoting the growth of the desired organism

Crystal or Gentian Violet
Basic or Carbol Fuchsin
Bile Salts
Sodium Desoxycholate

Inhibit growth of Gram-Positive Microorganism (4)

> Potassium Tellurite
> Sodium Azide

Inhibit growth of Gram-Negative Microorganisms (2)

> Alcohol
> Chloral Hydrate

Prevent Swarming of Proteus

Hektoen Enteric (HE) Agar
MAC
Xylose Lysine Deoxycholate (XLD)
Bismuth Sulfite Agar (BSA)
Mannitol Salt Agar (MSA)
Thayer Martin Agar (TMA)
Salmonella-Shigella Agar (SSA)
TCBS

examples of selective media (8)

Lysine, Lactose, Xylose and Sucrose

Xylose Lysine Deoxycholate (XLD)
Components:

▪ 0.25% Sodium Desoxycholate →inhibits gram-positive bacteria
▪ Phenol Red→ pH indicator
▪ Ferric Ammonium Citrate→ H2S indicator
* Salmonella→ colonies are red with black center

▪ ____ →inhibits gram-positive bacteria
▪ ____→ pH indicator
▪ ____ → H2S indicator
* ____ → colonies are red with black center

COLUMBIA CAN WITH BLOOD

three peptone sources and 5% defibrinated sheep blood

Colistin (C) and Nalidixic Acid (NA)

_____ → suppress the growth of most gram-negative organisms

PHENYLETHYL ALCOHOL (PEA) AGAR

sheep blood agar supplemented with phenylethyl alcohol to inhibit the growth of gram-negative bacteria

THAYER-MARTIN

enriched Chocolate Agar with supplement B or Isovitale X

▪ Colistin → inhibit gram-negative bacteria
▪ Vancomycin → inhibit gram-positive bacteria
▪ Nystatin → inhibit yeast

Antibiotic Components of thayer-martin:
▪ ___ → inhibit gram-negative bacteria
▪ ____ → inhibit gram-positive bacteria
▪ ____→ inhibit yeast

MODIFIED THAYER-MARTIN AGAR

chocolatized blood + antibiotics

Neisseria gonorrhoeae and Neisseria meningitides

specific bacteria used in modified thayer-martin agar (2)

▪ Colistin → inhibit gram-negative bacteria
▪ Vancomycin → inhibit gram-positive bacteria
▪ Nystatin → inhibit yeast
▪ Trimethoprim Lactate → inhibit Proteus spp.

Antibiotic Components of Modified Thayer-Martin Agar:
▪ ____→ inhibit gram-negative bacteria
▪ ____→ inhibit gram-positive bacteria
▪ ____→ inhibit yeast
▪ ____ → inhibit Proteus spp.

TRANGROW MEDIUM

Thayer-Martin with glucose, 2% agar, Trimethoprim Lactate incorporated in bottle

MARTIN-LEWIS AGAR

substitute Anisomycin for Nystatin and higher concentration of vancomycin

NEW YORK CITY MEDIUM

Modified Thayer-Martin with substitution of Amphotericin B for Nystatin

GENTAMICIN BLOOD AGAR

OTHER SELECTIVE MEDIA:
for Streptococcus

BACITRACIN CHOCOLATE AGAR

OTHER SELECTIVE MEDIA:
for Haemophilus

BLOOD AGAR PLATE WITH AMPICILLIN

OTHER SELECTIVE MEDIA:
for Aeromonas

SELECTIVE AND DIFFERENTIAL MEDIA

Used in the primary isolation of enteric Gram-Negative bacteria

1. Endo Agar
2. XLD Agar - Xylose Lysine Deoxycholate
3. MAC Agar - MacConkey agar
4. EMB Agar - Eosin methylene blue

examples of selective and differential media

SPECIAL MEDIA

Isolate bacteria with a specific growth requirements

Specially prepared to support the growth of specific microorganisms

1. Middlebrook 7H-10 Agar - M. tuberculosis
2. Fletcher Medium – Leptospira
3. “W” or Winsconsin Medium - Brucella
4. Bordet-Gengou Agar - Bordetella pertussis
5. Thayer Martin - Nesseria
6. MacBride - Listeria monocytogenes
7. Dieudonn’s Medium - Vibrio cholerae

1. ______ - M. tuberculosis
2. ______ – Leptospira
3. ______ - Brucella
4. ______ - Bordetella pertussis
5. ______ - Nesseria
6. ______ - Listeria monocytogenes
7. ______ - Vibrio cholerae

LOWENSTEIN-JENSEN
inspissation

Protein rich medium composed of whole egg and malachite green and supports the growth of Mycobacteria

Sterilization: ______

THIOSULFATE-CITRATE-BILE SALTS-SUCROSE (TCBS) AGAR
Boiling

Selective for the isolation of Vibrio
“Special Medium”
Sterilization:

1. Weighing - weigh the different ingredients then place in clean, dry containers
2. Dissolving - add the exact amount of solvent to the ingredients and then dissolve by heating
3. Titration - adjustment to the right pH: 7.2-7.4
4. Distribution - distribute in test tubes
5. Sterilization

1. ______- weigh the different ingredients then place in clean, dry containers
2. _____- add the exact amount of solvent to the ingredients and then dissolve by heating
3. ______- adjustment to the right pH: _____
4. ______ - distribute in test tubes
5. ______

PLATED METHOD

1. Weighing
2. Dissolving
3. Titration
4. Sterilization
5. Distribution

SPECIMEN CONSIDERATIONS FOR INOCULATION OF MEDIA

• Sterile body fluids, pus, urine and sputum > Inoculated directly into the selected media
• Specimens received on swabs > Can be inoculated directly into the culture media

Streaking

most common manner of inoculation

Overlapping inoculation

used for antimicrobial sensitivity test

Liquid Medium

with the use of sterile Pasteur pipet
> inoculate by shaking a previously heated wire loop or needle

Slant Medium

with the use of a wire loop or needle, transfer the inoculum to the bottom of the slant and streak in a zig-zag manner across the entire surface toward the mouth of the tube

Butt Medium

just stab the medium with an inoculating needle

Butt/Slant Medium

inoculate the butt first by stabbing the needle to the bottom of the medium and then streak the surface in a zig-zag manner toward the mouth of the tube

Streak Plate Technique

plate media to isolate organisms in pure culture

Pour Plate

plate media to determine the approximate number of viable organisms in a liquid such as water, milk, urine or broth culture

Streak-Pour Plate

plate media for studying hemolysis

4+

MANNER OF REPORTING (GRADING) OF GROWTH ON PLATE
many,heavy growth; growth is up to the fourth quadrant

3+

MANNER OF REPORTING (GRADING) OF GROWTH ON PLATE
moderate growth; growth is up to the third quadrant

2+

MANNER OF REPORTING (GRADING) OF GROWTH ON PLATE
few or light growth; growth is in the second quadrant

1+

MANNER OF REPORTING (GRADING) OF GROWTH ON PLATE
rare growth; growth is in the first quadrant only