2. Definition of Media:
• The medium that used to culture
microorgamis the one trying to
isolate.......Nutrients may be added to
make medium higher with protein or
sugar..
• Ph added for differentiation microbes
based on thier biochemical reactions..
• Mother ingredients (growth factors...
Nacl... ph buffers..) For microbes
metabolism.
3. Classifications of media according
to consistency:
• 🌸🌸 Solid media
• Defination /
• An inert solidfying agent it
has a physical structure and
allow bacteria to grow in
physically informative..
• (ex/ agar media..)
•
• Function /
• isolate bacteria
• determination colony
characteristics.
• 🌸🌸 Liquid media
• Defination/
• It contains specific nutrients
• (ex / broth media..)
•
• Function /
• Propagation of large
numbers of organisms..
• Fermentation studies (ex/
suger fermentation test)
4. Classification of culture media
according to composition :
• Synthetic chemical defined medium /
• Prepared from purified ingredients and their composition is known.
• They may be simple or complex depending on their supplement incorporated on it.
• Non-synthetic chemically
undefined medium/
• It contains at least one
component that not purified or
compeletly characterized.
• Often these media digest proteins
from various organisms sourses
• (Ex/ nutrient broth from culture of
yeast)
• There is simple non -synthetic
medium capable of meeting
nutrients requirements of
organisms requiring some growth
factors as in complex non -
synthetic medium that support
growth
5. 1-Broth culture madium :
•Defination/
• Are liquid cultures used to grow bacteria in
laboratories.
• To create a broth culture, ascientist begins with
asterile liquid growth medium.
• The medium is inculated with bacteria and
placed in an incubator at the appropriate
temperature.
• After a certain time the broth becomes cloudy
from the increased number of microbes..
8. Procedures of broth media
prepation..
• 1_ pour 500 ml of distilled water to the medium and added,
peptone _ beef extract _ sodium chloride..
• 2_ mix the content and heat it with continuous agitation to dissolve
the consistuents.
• 3_ add more distilled water to the medium and make the volume
1000 ml
• 4_ check the phone of the solution using pH strip _it should be 7,2 .
• 5_ mix the content well and apply the non obsorbent cotton plug to
the flask
• 6_ autoclave the contents at 121C and 15 psi pressure for 15
minutes.
• 7_ allow the content to cool and inculate specimen to be cultured.
9. Precaution to be taken in broth
media preparing..
• 1_ carefully measure the peptone and beef
extract and sodium chloride.
• 2_ maintain the pH of the medium..
because a single drop can change the pH of
solution.
• 3_ store the medium at low temperature.
• 4_ acid and alkali are corrosive to skin.. So
handle with it carefully...
10. 2_ Agar culture medium
• Defination/
• APetri dish that contains agar as a solid
growth medium plus nutrients used to
culture microorganisms
• Sometimes selective compounds are
added to influence growth such as
antibiotics.
11. Types of agar Media..
• 1_ blood agar.
• _contains blood cells from animals , bacteria..
• 2_ cluria bet
• 3_ macconkey agar.
• 4_ miller's LB agar.
• 5_ Neomycin agar.
• 6_ No nutrient agar.
• 7_ nutrient agar.
• 8_ sabouraud agar.
• 9_ thayer _ Martin agar.
• 10_ tryptic soy agar.
• 11_XL D agar.
•
12. Materials that used in agar media
preparation...
• 1_ 0, 5% peptone.
• 2_ 0,3 % beef extract / yeast extract.
• 3_ 1,5 % agar , this gives the mixture solidity.
• 4_ 0,5 f sodium choliride.
• 5_ distilled water.
• 6_ pH adjusted to neutral (6,8) at 25 C.
13. Procedures of agar media
preparation..
• 1_ add 28 g of nutrient agar powder in 1 liter of
distilled water.
• 2_ heat the mixture while stirring to fully
dissolve all components.
• 3_ autoclave the dissolved mixture at 121
degree celsieus for 15 minutes.
• 4_ once the nutrients agar has been autoclaved
allow it to cool but not solidfying.
• 5_ pour nutrients agar into each plate and leave
plates on the sterile surface until the agar has
solidified.
14. Precautions to be taken in agar media preparaing..
• 1_ never leave the culture dish open.
• 2_flame a loop or needleto red _hot just prior
the use.
• 3_Burning off any organic material.
• 4_cool the instrument by touching the sterile
agar or liquid surface prior touching a sterile.
• 5_ re_sterile the instrument after doing the
procedure.
•
16. Quality Control
Checking of different parameters of media such as gro
wth supporting characteristics, physical characteristics
, gel strength and batch contamination can help to ass
ess their quality .
Definition:-
• When to perform media QC?
– In-coming raw material (base media powder)
– After manufacture
– After additional treatment, such as irradiation
– To assess delivery (heat shock and cold shock)
– To assess changes to storage conditions (e.g. Cold store )
– As part of shelf-life studies (the expiry time)
17. The color of the media should be examined and a decisi
on made as to its correctness, as well as an examination
for any crystal formations or variations in color (for agars
). The containers of media should be thoroughly examin
ed for cracks or defects, and all defective units discarde
d
Physical parameters
– Solid media: excessive bubbles or pits, unequal filling of plates (u
niform levelling), cracked medium in plate, gel strength and freez
ing or crystallization
– Liquid media: Clarity of broth
– All media: pH value of the medium (pre- and post-sterilisation)(p
H measurement must be conducted at room temperature unless
specific allowance is made for the temperature)
18. Media prepared must be stored under appropriat
e and controlled conditions and used within esta
blished expiry dates. The compounding of the me
dia must be controlled to ensure the media is pre
pared correctly. Agar media must be pre-warmed
to dissolve the agar prior to sterilization, but not
heated so extensively as to damage any heat-labil
e components. The sterilization procedure also m
ust be under control. Normally this means using
a validated autoclave cycle (and load configuratio
n) shown to hold the media at 121oC for 15 minut
es . Each batch of media should be clearly labele
d to allow for unambiguous audit of each stage of
preparation.
19. • Control of Storage Conditions:-
• Media Quarantine and Release:-
• The laboratory must have some procedures in pl
ace to prevent unqualified media from entering th
e testing process. This ideally would be a separat
e storage room from that used to store qualified
media, but may also be accomplished through ta
gging the quarantined material and placing it in a
clearly identified area within the same room. All q
uality control checks on the quarantined media sh
ould be completed before its documented release
for general use. Storage conditions of the quarant
ined media should match those of the released m
edia
20. Media Storage and Expiry:-
Media should always be stored under controlled con
ditions to ensure its quality through to the expiry dat
e. Factors to be evaluated in these controlled conditi
ons include:
-Temperature
-Container (glass, plastic, container closure system,
etc)
-Humidity
-Light
21. • The time of incubation for some media is defined in the ph
armacopoeias (e.g. Microbial Enumeration Test)
• For others, user needs to defineTarget which is often:
– Up to 3 days for bacteria
– Up to 5 days for fungi
• Different storage conditions affect:
– Drying out (due to different humidity levels, which
can affect the water activity of solid media)
– Chemo-oxidation (due to physical factors like heat
)
– Photo-oxidation (from sunlight)
22. The Sterility Test states that:
“If prepared media are stored in unsealed cont
ainers, they can be used for 1 month, provided
that they are tested for growth promotion withi
n 2 weeks of the time of use and that color indi
cator requirements are met. If stored in tight c
ontainers, the media can be used for 1 year, pr
ovided that they are tested for growth promotio
n within 3 months of the time of use and that t
he color indicator requirements are met .
”
Raw materials: copper ions, conductivity and pH
The quality of Petri dish used for pouring of media is also an important factor. Normally petri dishes are ethylene oxide (EtO) sterilized or gamma irradiated. If EtO sterilized they should be then checked for residual EtO toxicity, as this may affect the growth of the microorganisms
Sterilization of the media plays an important role in the quality of the media. Generally autoclaving is carried out for sterilizing the media. However, the time of autoclaving and the quantity of media sterilized should be closely regulated.[1] Heat treatment of complex culture media may result in its nutrient destruction either by direct thermal degradation or by reactions between the components. Therefore, it is very important to optimize the heating process to minimize heating damages. The suggested cycle is stage 1:20-121°C, stage 2: < 100-121°C, stage 3:121-121°C and stage 4:121-80°C.
Expiry is important
Growth index is a bit iffy
Limit of detection cannot be determined but EM media should be able to detect low counts
A paradox for media used at Grade A….maybe frequency of counts is best
