MICROPARA LAB: MICROBIOLOGY MOD SEM

AND PARASITOLOGY 4 1
MODULE 4: PREPARATION OF CULTURE MEDIA

CULTURE • “growth media” ✓ ensure you are wearing appropriate protective

MEDIA
• a nutrient material prepared for the clothing
growth of microorganisms ✓ only use sterile glassware, equipment, media, and
reagents
INOCULUM • microbes that are introduced into a ✓ handle only one microbial culture at a time.
culture medium to initiate growth ✓ test tube cultures: hold cultures at an angle after
you remove the lid
CULTURE • microbes that grow and multiply in or
✓ sterilize the outside of the culture tube using a
on a culture medium
bunsen burner flame
CRITERIA TO GROW A CULTURE OF CERTAIN BACTERIA ✓ plated cultures: hold the petri dish lid at an angle
a. right nutrients after you remove the lid
b. sufficient moisture ✓ microbial culture: work quickly and carefully in an
c. properly adjusted pH environment that has minimal distractions
d. suitable level of oxygen ✓ never take cultures outside of the laboratory
e. sterility ✓ notify the laboratory supervisor immediately of
f. incubation at proper temperature
any spills

RESEARCH FINDINGS
ASEPTIC TECHNIQUES
LOOPS & • sterilize by inserting it into a
NEEDLES bunsen burner or incinerator
WORK AREA
flame until it is red-hot
✓ before and after use, disinfect all work surfaces
CULTURE TUBE • prior to inserting a cooled loop
with 70% ethanol or an appropriate disinfectant FLAMING & or needle into a culture tube,
✓ maintain an uncluttered work space INOCULATION the cap is removed and the
✓ ensure that you have all necessary supplies before mouth of the tube may be
beginning an experiment flamed
✓ work may be performed in a thoroughly sterilized → broth tube
biosafety cabinet → agar slant
✓ do not open windows or use fans that circulate → stab culture
outside air
✓ frequently clean water baths used for thawing or PERI PLATE INOCULATIONS
warming media or solutions • the plate cover is raised and held diagonally over
✓ routinely sterilize incubators used for microbial the plate to protect the surface from any
propagation contamination in the air
• the loop containing the inoculum is then streaked
HANDLING MEDIA
gently over the surface of the agar
✓ before and after use, sterilize the outside
• the cover is replaced and the loop is flamed
container of all media and reagents with 70%
ethanol
FINAL FLAMING OF THE LOOP/NEEDLE
✓ aliquot sterile solutions into smaller volumes
whenever possible • loop or needle is flamed to destroy any organisms
✓ avoid pouring sterile liquids from one container to that remain on these implements and is returned to
another its receptacle storage
✓ never mouth pipette
✓ always use sterile glass or disposable plastic CLASSIFICATION BASED ON PHYSICAL STATE/CONSISTENCY
pipettes to work with liquid media SOLID MEDIA • 1-3% agar
✓ do not open sterile media, petri dishes, or pipette • unbranched polysaccharide
containers until you are ready to use them obtained from the cell
• membranes of some species of
HANDLING MICROBIAL CULTURES red algae
✓ before working with media and microbial cultures, • 70% agarose + 30% agarapectin
wipe your work area with 70% ethanol or an • most used solidifying agent
appropriate disinfectant • not hydrolyzed by most bacteria,
free from growth promoting or

GIANNA ALEXIS LINGAD 1

 growth retarding substances
• used when solid medium is DIFFERENTIAL/INDICATOR MEDIA
desired for bacterial growth • contain compounds that allow some groups of
commercial use: thickener microorganisms to be visually distinguished by the
contained in: appearance of the colony or the surrounding
→ slants media
→ deeps examples:
→ petri plates
→ MacConkey’s agar
SEMI-SOLID • 0.2-0.5% agar → CLED agar
MEDIA • fairly soft → TCBS agar
• useful in demonstrating → XLD agar
bacterial motility and separating
motile from non-motile strains
TRANSPORT MEDIA
LIQUID BROTH • “broth”
• media prevent drying (desiccation) of specimen,
• bacteria grow uniformly maintain
producing general turbidity
• used when a large number of • the pathogen to commensal ratio and inhibit
bacteria have to be grown overgrowth of unwanted bacteria
example:
CLASIFICATION BASED ON NUTRITIONAL COMPONENT → stuart & amie’s media
→ cary blair medium
SIMPLE MEDIA • support most non-
fastidious bacteria → sach’s buffered glycerol saline
examples:
peptone water, nutrient agar ANAEROBIC MEDIA
COMPLEX MEDIA • made up of nutrients • need low oxygen content, reduced oxidation –
including extracts from reduction
yeasts, meat, or plants, or • potential and extra nutrients
digests of proteins from reduced by physical or chemical means:
these and other sources boiling the medium serves to expel any dissolved
• exact chemical oxygen in addition of:
composition varies slightly → 1% glucose,
from batch to batch → 0.1% thioglycollate,
→ 0.1% ascorbic acid
example: blood agar → 0.05% cysteine/red hot iron filings
SYNTHETIC/DEFINED • one whose exact chemical can render a medium reduced
MEDIA composition is known
PREPARATION AND PRESERVATION OF CULTURE MEDIA
CLASSIFICATION BASED ON FUNCTIONAL USE OR 1. care must be taken to adjust the ph of the medium
APPLICATION before autoclaving
BASAL MEDIA 2. dehydrated media are commercially available and
• routinely used simple media having carbon and must be reconstituted as per manufacturers’
nitrogen sources that boost the growth of many recommendation
microorganisms 3. most culture media are sterilized by autoclaving
• general-purpose media, nonselective media 4. certain media that contain heat labile components
like glucose, antibiotics, urea, serum, blood are NOT
• peptone water, nutrient agar autoclaved
5. certain highly selective media such as wilson and
ENRICHED MEDIA blair’s medium and tcbs agar need NOT be sterilized
• used to grow nutritionally exacting (fastidious) 6. a representation from each lot should be tested for
bacteria performance and contamination before use. once
examples: blood agar, chocolate agar, loeffler’s serum slope prepared, media may be held at 4-5C in the
refrigerator for 1-2 weeks
SELECTIVE 7. certain liquid media in screw capped bottles or tubes
• designed to inhibit unwanted commensal or or cotton plugged can be held at room temperature
for weeks
contaminating bacteria and help to recover
pathogen from a mixture of bacteria
DETECTION OF CONTAMINATION
• increase of turbidity of the antibiotic-free

GIANNA ALEXIS LINGAD 2

 medium: color becomes cloudy
• distinct shape under the light microscope (high
power microscopy (400X)
BACTERIA • space between cells will appear
uniformly
• granular and may shimmer from
the movement of the bacteria
YEAST & • appearance of clumps, mats,
FUNGI budding (yeasts) and colonies on
the surface of the media (fungi)

CHANGE IN PH
BACTERIA • (+) aerobic bacteria: medium
becomes acidic = yellow
• (+) anaerobic bacteria: medium
become alkaline = pink
MEDIA w/ • bacteria: medium = yellow
PHENOL RED • fungi: medium = pink
YEAST & • medium becomes alkaline
FUNGI

GIANNA ALEXIS LINGAD 3