Culture Media Preparation Flashcards
– can also be prepared for fungi and bacteria
culture media
– virus can be grown in this
living cells
- the survival of microorganisms depend on
available nutrients
favorable growth environment
nutrient preparations that are used for growing microorganisms
media or culture media
microbes that grow and multiply or on a culture medium
* three physical forms of media
culture
- three physical forms of media
liquid or broth media
semisolid media
solid media
ex. Nutrient broth, tryptic soy broth, brain-heart infusion broth
liquid or broth media
– have agar (ex. Motility medium)
semisolid media
have agar (ex. Nutrient agar, blood agar)
solid media
complex carbohydrate (polysaccharide) extracted from seaweed
agar
a solidifying agent
agar
solid agar melts about
100 degrees c
liquid agar solidifies at about
42 degrees c
while in the liquefied state, semi solid and solid media can be poured into either a test tube or petri dish/plate
agar
type of medium (5)
broth
agar slant
agar deep
agar stab
agar plate
autoclaving container for
broth
agar slant
agar deep
agar stab
agar plate
culture tube (broth - agar stab)
~200ml culture bottle or EM flask (agar plate)
- types of Culture Media (2)
chemically defined or synthetic
Chemically complex or non-synthetic
composed of known amounts of pure chemicals
chemically defined or synthetic
used in culturing autotrophic microorganisms such as algae or non-fastidious heterotrophs
chemically defined or synthetic
composed of complex materials that are rich in vitamins and nutrients
beef extract, yeast extract, and peptones
chemically complex or non synthetic
- culture media preparation
- calculation and weighing
- mixing with water and heating
- sterilization
- fine-tuning (supplements and pH)
- dispensing
- packing and storage
- quantity control
- use of dispensed culture media
temperature higher than 50oC poured in media can cause this
condensates
used to prevent buildup of condensation
inverted media
- Sterilization of media and materials for microbial culture
Culture dishes
Test tubes
Flasks
Pipettes
Transfer loops
media
using an oven, this kind of sterilization is carried out at 150oC to 180oC for 1 to 4 hours
dry heat sterilization
Can be used for glassware, fixed oils, thermostable powders
dry heat sterilization
sterilization using this requires higher temperatures and longer
dry heat sterilization
heat transfer is slow, small volumes of oil and thin layers should be used
dry heat sterilization
in the presence of moisture, microorganisms are destroyed at a lower temperature than in dry heat
moist heat sterilizaton
autoclave is using heat + pressure
moist heat sterilization
180 degrees at 150 psi
121.0 °C / 249.8 °F at 15 lbs / square inch (psi) for 15 minutes
Method of choice when product can withstand treatment (heat stable)
dry heat sterilizaton
- Steam sterilization applications
Solutions sealed in containers ampules, vials
Media
Bulk Solutions
Glass wares (ex. tubes with media)
Surgical Dressing
Instruments
- Advantages of steam sterilization:
Rapid, Inexpensive, Effective, Large volumes
- Disadvantages of steam sterilization:
Cannot be used for oily preparation (oil base ointment)
Cannot be used for moisture sensitive preparations
where culture medium is sterilized in
autoclave
varies from original value when subjected to heat sterilization (can increase/decrease_
pH of media
most bacteria grow best at this pH
pH 6.5-7.5
– pH should be adjusted after this
sterilization
very little grow at this pH
pH 4
may grow in pH as low as 1
acidophiles
- must be adjusted after sterilization if the pH of medium strays too far from neutral.
pH
- Preparation of media from commercial dehydrated products
Each bottle of dehydrated medium has instructions for preparation on its label
Nutrient agar 23g/1000ml
formula to find the g needed
=(how many g (how many mL))/1000mL
n order to make 400 mL of medium with a concentration of 15 g/L, how much medium powder would you weigh in grams
6g
- In order to make 650 mL of medium with a concentration of 20 g/L, how much medium powder would you weigh in grams?
13g
- To fill up 10 agar plates that contains 20 mL of medium with a concentration of 36 g/L, how much medium powder would you weigh in grams?
7.2g
- Preparation of Nutrient Broth (NB), a chemically complex medium
Prepare 300 ml of NB
Weigh appropriate NB powder and dissolve in 300 ml dH2O
Check initial pH of medium
Divide contents into 3 flasks
= 100 ml in each
- Sterilization of media:
1st flask – in autoclave (121⁰C, for 15 mins.)
2nd flask – in oven (170 ⁰C, for 1 hr.)
3rd flask – no sterilization
- Preparation of Glucose Minimal Salth Broth (GMSB)
Weigh media powder appropriate for 300 ml.
Combine media and dH2O in flask.
Check initial pH of medium
Divide contents into 3 flasks
= 100 ml in each
Sterilize. - autoclave
retain coloration and structure of bacteria during staining
mordant (gram’s iodine)
helps in the removal of primary stains from bacteria
decolorizer
escherichia coli
shape
gram positive/negative
Gram-negative, rod-shaped
Bacillus subtilis
gram positive/negative
gram positive, rod-shaped
Proteus vulgaris
gram/positive negative
gram-negative, rod-shaped
Serratia marceschens
gram/positive negative
rod-shaped, Gram-negative
Pseudomonas aeruginosa
gram/positive negative
rod-shaped Gram-negative rod
Staphylococcus aureus
gram positive/negative
Gram-positive, spherical or cocci-shaped
dye for positive staining
methylene blue
is a simple tool used primarily by microbiologists to take and transfer a small sample (inoculum) of a microorganism culture
inoculation loop
view experiment results on gc
+1
20 petri dishes will be used that will have 20mL NA/plate
28.0g/1000mL
compute for the grams
(28g)(20x20)/1000mL
11.2g
Normal Saline Solution abbreviation
SNSS
20 test tubes will be filled with 10 mL per test tubes
How much NaCl will be used to make 0.9% SNSS soln?
wt% = g solutes/ml of soln *100
0.009(250) = g
g= 2.25g
