CULTURE MEDIA

Medical Laboratory Science Batch 2022

 CULTURE MEDIA Medical Laboratory Science Batch 2019

Cultivation
• The process of growing microorganisms in culture by taking bacteria
from the infection site (in vivo environment)

• growing them in an artificial environment in the laboratory (in vitro)

 CULTURE MEDIA Medical Laboratory Science Batch 2019

Agar
• Agar or agar-agar is a gelatinous
substance derived from a
polysaccharide that accumulates in
the cell walls of agarophyte red algae

• Gelidium amansii

Microbial growth does not destroy the gel

structure because most microorganisms
are unable to digest agar
 CULTURE MEDIA Medical Laboratory Science Batch 2019

INGREDIENTS COMMONLY USED IN

MEDIA
• Beef Extract. It contains amino acids, vitamins and
minerals.
• Peptone. Protein is converted into proteoses, peptides
and amino acids by means of enzymatic hydrolysis.
• Yeast Extract. Contains amino acids, nucleic acids,
vitamins and other growth factors.
 CULTURE MEDIA
Medical Laboratory Science Batch 2019

General Steps in Preparation of culture Medium

• Weighing – weigh the different
ingredients and then place in clean, dry
containers
• Dissolving – add the exact amount of
solvent to the ingredients and then
dissolve by heating
• Titration – adjustment to the right pH
(ph 7.2)
• Sterilization - Autoclave/Oven
• Distribution – distribute in petri plates
 CULTURE MEDIA
Medical Laboratory Science Batch 2019

General Steps in Preparation of culture Medium

• Weighing – weigh the different
ingredients and then place in clean, dry
containers
• Dissolving – add the exact amount of
solvent to the ingredients and then
dissolve by heating
• Titration – adjustment to the right pH
(ph 7.2)
• Distribution – distribute in petri plates
• Sterilization - Autoclave/Oven
 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media

According to consistency/physical
state
• Liquid /broth – no agar/solidifying
substances (Tryptone Soy Broth (TSB)
, Thioglycolate)
 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media

Liquid /broth
Tryptone Soy Broth
A general purpose nutritious broth capable of growing a
wide range of bacteria

• Non-fastidious and fastidious

Composition:
• casein peptone
• nitrogen, vitamins, minerals, and amino acids
• Glucose
 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media

Liquid /broth
Tryptone Soy Broth
• Boiled – to drive off oxygen

Composition
• Casein peptone
• Sodium thioglycollate and L-Cystine (Lowers oxygen tension)
• Glucose (dextrose)
• NaCl – prvovides essential electrolytes for transport and osmotic balance
 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media

Semi-solid (0.5 – 1%) Solid (2 – 3%)

Sulfide Indole Motility (SIM) Blood Agar Plate

 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media

According to Use
• Simple/supportive/basal
• allows most non-fastidious organism to
grow (Nutrient Agar)

• Enrichment medium
• liquid medium that favors the growth of 1 or
more organism (Alkaline Peptone Water)

Nutrient Agar APW

 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media

According to Use
• Enriched Medium – solid form, contains
nutritive requirements. (Blood Agar Plate
(BAP), Chocolate Agar Plate (CAP)

Blood Agar Plate Chocolate Agar Plate

 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media

According to Use
• Selective media – grows particular organism
• Contains inhibitors

Inhibitors for Gram-positive organisms Inhibitors for negative organisms

• Gentian violet • Potassium tellurite
• Sodium deoxycholate (bile salts) • Sodium azide
• Basic fuchsin
 CULTURE MEDIAMedical Laboratory Science Batch 2019

Classification of culture media

According to Use
• Selective media – grows particular organism
• Contains inhibitors

Prevents swarming of Proteus

• Alcohol
• Chloral hydrate
 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media Selective Media

Lowenstein Jensen
medium: Increase Mueller Tellurite Medium
protein, EGG BASED
Inhibitor: K tellurite
Inhibitor: malachite green Colonies: gray – black
colonies

Mycobacterium tuberculosis Corynebacterium diptheriae

 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media Selective Media

Selective for Neisseria gonorrheae

Thayer Martin Modified Thayer Martin (MTM)

• Vancomycin – inhibits gram (+) • Vancomycin + Colistin + Nystatin
• Colistin – imhibits gram (-) • Trimetophrim lactate – inhibits
other than itself swarming of Proteus
• Nystatin – inhibits fungi
 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media Selective Media

Selective for Neisseria gonorrheae

Martin Lewis New York City Agar

• Vancomycin+Colistin + • Vancomycin+Colistin+Trimetophrim
Trimetophrim lactate lactate
• Anisomycin – inhibits fungi • Amphotericin B – inhibits fungi
 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media Selective/Differential

for Staphylococcus sp. Vibrio sp.

• Medium: MSA Medium: TCBS: Thiosulfate

• Inhibitor: 7.5 % NaCl Citrate Bile Salt Sucrose Agar
CHO: Sucrose
• CHO: mannitol pH indicator: Bromthymol
• pH indicator: Phenol red blue
• Mannitol fermenter: “yellow halo” SF: yellow (Cholerae,
alginolyticus)
• Non Mannitol Fermenters – Pink nSF: Green (parahemolyticus)
 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media Selective/Differential

for Enterobacteriaceae
Eosin – Methylene Blue (EMB) agar
MacConkey (MAC) Agar
Inhibitor: Crystal violet and Bile CHO: Lactose
salts LF: pink – purple
CHO: Lactose nLF: Colorless

pH indicator: Neutral Red E. coli: Green Metalic Sheen

LF: Pink Klebsiella: Pink mucoid colonies
Enterobacter: Pink colonies with
nLF: Colorless dark center. “FISH EYE” colony
 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media Selective/Differential

for Enterobacteriaceae
Hektoen Enteric Agar (HEA) Salmonella Shigella Agar (SSA)
Inhibitor: bile salt CHO: lactose
CHO: Lactose, Sucrose, pH indicator: Neutral Red
Salicin H2S indicator:(Fe+NH4 Citrate)
pH indicator: Bromthymol Salmonella: H2s (+), colorless w/
blue black center
H2s indicator: (Fe+NH4
Shigella: H2S (-), Colorless, w/o
Citrate)
black center
 CULTURE MEDIA Medical Laboratory Science Batch 2019

Classification of culture media Selective/Differential

Selective (CNA), Differential (BAP)

Beta – Complete hemolysis of red blood cells Alpha – incomplete hemolysis Gamma – No hemolysis
 CULTURE MEDIA Medical Laboratory Science Batch 2019

Medium for Susceptibility Testing

Mueller-Hinton Agar

Agar depth – 4mm

pH: 7.2 – 7.4

Distance between 2 disks: 24 mm

 CULTURE MEDIAMedical Laboratory Science Batch 2019

Special Culture Media

Specially prepared to support the growth of specific microorganisms
Examples:

• Middlebrook 7H-10 agar - M. tuberculosis

• Fletcher medium - Leptospira
• W medium - Brucella
• Bordet-Gengou agar - Bordetella pertussis
• MacBride - Listeria monocytogenes
CULTURE MEDIA
Medical Laboratory Science Batch 2019