media prep Flashcards
(30 cards)
what is agar made from? actual thing and compounds
prepared from seaweed.
Made up of two polypeptides: agarose and agaropectin which vary in ratio depending on the type of seaweed/algae
resposibilities of agarose and agaropectin
agarose is necessary for the high strength gelling properties and agaropectin is used to give the viscous properties.
necessary characteristics of agar
- soluble in hot water but insoluble in cold
- mustbe free from: toxic compounds, dead baceria, free sugars, impurities, insoluble fats, live termophilic organisms
- does not alter the final pH
- cannot be heated more than 2 times or it will be semisolid
3 types of agar (not plates just depending on what its used for)
- bacteriological agar used in culture media
- processed bacteriological agar for susceptibility testing
-technical grade agar for immuno-electrophoresis and gel difusion studies
types of water soluble proteins in media
obtained from lean meat, casein, heart muscle or soya flour.
- soya peptone used in antibiotic production
- casein hydrolysate used in antibiotic and vaccine production
- malt extract is used in yeast and mould media
peptones, proteoses (stages of hydrolytic breakdown of proteins before reaching amino acids) and amino acids
characteristics of peptones in media
- moisture content of less than 5%
- pH 5-7
- hygroscopic (absorb from air)
- support growth of non fastiduous bacteria
- have no fermentable carbs
- low content of contaminating bacteria
- very low content of copper
key ingerdients of culture media
- peptones, proteoses and a.a for nitrogen and nutrients
- metals and minerals (Ca, Mg, Fe, phosphates)
- carbs fr energy
- phosphates and acetates as buffering agents
- indicators
-selective agents - gelling agent - agar
- others such as growth factors
treatment of horse/sheep blood for culture media
- collected aseptically
-defibrinated immediately - potassium oxalate (10mls per litre) and or sodium citrate (60mg or 10mls per litre )may be added as anticoagulants
- may be haemolysed chemically via saponin or mechnically by freezing and tawing (therefore not allowed to be frozen)
- stored at 2-4 degrees, expiry at 15 days.
treatment of horse blood serum for blood agar
-sterilized by filtration or taken under aseptic conditions
-stored at 3-5 degrees or deep freezed and thawed before use
-free from antibiotics and bacteria
water used for media prep
deionized or destilled
pH of 5.5-7.7.
copper material cannot be used for distillation.cannot contain Cu as it is toxic to bacteria
conductivity of less than 15 siemens
caution when autoclaving medium with carbs
autoclaving at 121 would lead agar becoming dark brwon due to caramelization. add carbs aseptically after autoclaving
about bile salts in media
derived from ox or pigs
salts of sodium taruocholate and sodium glycocholate
used to inhibit non enteric bacteria (like commensals)
precautions on dealing with powder culture media and its preparation (not the method ust with apparatus and weighing etc)
- hygroscopic so avoid leaving open (absorbs water from air)
- sensitive to moisture, heat, light and drastic changes in temp
- imp to store in correct storage conditions
- clean, dry spatula used every time for weighing
- vessel has to be at least twice the size of the final volume
- opne bottle away from droughts and moisture
- avoid clouds of dust as they are harmful to inhale.
- glassware only used for media prep and not alcohol or stains as these may inhibit batcerial growth.
types of petri dishes
found as 90ml or 140ml plates.
- no vent: no gas exchange
- single vented: slight gas exchange
- triple vented: gas exchange
types of McCartney Bottles
- 28mls universal container (wide mouth)
- 7mls McCartney container ‘Bijoux’ (used for carb studies or transport media)
- 14mls McCartney Container (used for slants)
- 28mls McCartney Container + self healing rubber to inject needle if necessary.
storage of screw capped bottle of basal media
- stored for up to 6 months
- at 12-16 degrees
-away from light
storage of agar plates
at 2-8 degrees
- away from light
- do not freeze
- sealed container to avoid loss of moisture
filter sterilization
carried out with 0.22 micrometers: used to sterilize delicate supplements inactivated by autoclaving (used for growth supplements, antibiotics and carbs).
stages of the sterilization cycle
- Chamber heat up time 20-100 degrees
- Heat penetration time of medium container 100-121 degrees
- Holding time at prescribed temperature 121-121 degrees
- Cool down 121-80 degrees
remelting solid media
autoclavable media can be remelted by autoclaving–> this leads to overheating which causes pH drift, darkening, precipitation, poor gel strength and reduced bacteriological performance.
free steaming is preferred using a Koch steamer. It may not be remelted more than twice.
steam for 20-25 mins per 100mls or autoclave 5-15 minutes at 121 degrees
possible faults in media prep
wrong pH value
turbidity, precipitation
darkening
poor bacterial growth
soft gel
causes for the wrong pH value
- pH test carried above 25 degrees
- overheating through prolonged sterilization
- incomplete solution of medium
- poor quality of medium
- glassware has to be of suitable and appropruate quality: avoid soda glass and neutralize where necessary
- check that water pH is not low or high
do not adjust media pH before autoclaving
causes for turbidity and precipitation
ingredients would’ve precipitated out of solution
- poor quality water
- overheating through prolonged sterilisation
- pH value incorrect
- incomplete solution
causes from darkening
-pH drift
- overheating
- incomplete solution of medium
