Preparation of media

Methods of sterilization of
media &
equipment/glassware

BIO 3213
FACULTY OF NATURAL SCIENCES
UNIVERSITY OF GUYANA
OUTLINE
 Dehydrated culture media

 Preparation of culture media

 Checking the pH of a culture medium
 Sterilizing culture media
 Sterility testing
 Performance testing
 Control species
 Labelling & storage of culture media & additives
 Sterilizing glasswares in a hot air oven

 How to dispense culture media

 Dispensing sterile media into petri dishes
 Dispensing & solidifying high protein media (inspissation)
Dehydrated culture media
Dehydrated culture media
 Ready-made

Used to ensure good performance & reproducibility

Less expensive than buying the individual chemical

constituents

 Some chms may also be difficult to obtain, not available

in small amounts & may have a short shelf-life

Hygroscopic

 If exposed to moisture , it rapidly becomes unfit for use

 A hard mass is formed which alters the chemical &
microbiological properties of the medium
Dehydrated culture media - Precautions
To prevent deteriorating & having to discard it
before it is finished

Weighing the medium rapidly & tightly capping

the bottle ASAP after removing the approx.
amount
- Do not return small amounts of medium to the
stock bottle

 Sealing the cap of the container with adhesive

tape or seal the container in an airtight plastic bag

Storing media in the coolest driest place available

& always out of sunlight
Preparation of culture media
Preparation of culture media
Prep details & QA of all culture media used in the lab must be
included in SOPs
A record MUST BE kept of stock items, sources of materials & the
dates when different media are prepared

Prepare media made from dehydrated products in a damp-free

environment

Once the ingredients are weighed, do not delay in making up

the medium. Follow exactly the manufacturer’s instructions

Use completely clean glassware, plastic or stainless steel

equipment that has been rinsed in pure water
Preparation of culture media
The container in which the medium is prepared should have a
capacity of at least twice the volume of the medium
being prepared

Use distilled water from a glass still. Deionized water can

also be used providing the exchange resins do not contain
substances inhibitory to bacteria

Add the powdered or granular ingredients to the water & stir

to dissolve
 Do not shake but mix by stirring or by rotating the
container
Preparation of culture media
When heating is required to dissolve the medium, stir while
heating & control the heat to prevent boiling & foaming
which can be dangerous & damage the medium (DCA or TCBS
agar)
 Overheating a medium can alter its pH, nutritional & gelling
properties

Autoclave a medium only when the ingredients are completely

dissolved

Dispense medium in bottles or tubes in amounts convenient for

use
 Know the length of time prepared media can be stored w/o
deteriorating
Checking the pH of a culture medium
The pH of most culture media is near neutral except alkaline
peptone water

Use narrow range pH papers or a pH meter

A fluid medium can be tested by dipping a narrow range pH

paper into a sample of the medium when it is at RT & comparing
the colour of the paper against the pH colour chart provided

An agar medium can be tested by pouring a sample of the molten

medium into a small beaker or petri dish & when it has solidified,
laying a narrow range pH paper on its surface
 The colour of the paper is then compared against the pH colour
chart
Checking the pH of a culture medium
The pH of a dehydrated medium should not require
adjustment providing it has been prepared correctly using
pure water & clean equipment, and it has not been over-
autoclaved

The pH of other media should be adjusted as directed in the

method of prep
 Minor adjustments should be carried out using 0.1 mol/l
(N/10) sodium hydroxide when the medium is too acid & 0.1
mol/l (N/10) hydrochloric acid when too alkaline
Sterilizing culture media
The following methods are used to sterilize culture media:
– Autoclaving
– Steaming at 100 C
– Filtration
Sterilizing culture media - Autoclaving
For majority of culture media

Destruction of bacterial endospores as well as

vegetative cells

A medium should be sterilized at the correct temp

& for the correct length of time as instructed in
the method of prep

Underautoclaving: unsterile medium ~ discarded

Over-autoclaving : precipitation, alteration of pH &

the destruction of essential components in a
medium
Sterilizing culture media - Steaming at 100 C

Used to sterilize media containing

ingredients that would be broken
down or inactivated at temp
over 100 C e.g. Cary-Blair transport
medium

Used to re-melt previously

bottled sterile agar media

Can be performed in an autoclave

with the lid left loose or in any
form of steam sterilizer (Arnold or
Koch steamer)
Sterilizing culture media - Steaming at 100 C

The bottles of media with loosened caps

are placed on perforated trays above the
boiling water

After sterilization & the medium has

cooled, the bottle tops are tightened

Steaming times vary according to the

type of medium, e.g. 15 mins for Cary-
Blair medium
Sterilizing culture media - Filtration
Removing bacteria from fluids

Used to sterilize additives that are heat-sensitive

or less stable substances that need to be added to
a sterile medium immediately before it is used
 E.g. serum & solutions containing urea & certain
CHO

Different types of filters can be used including

those made from sintered glass or inert
cellulose esters
 Cellulose filters are called membrane filters
 Preferred b/c they filter more rapidly, do not
affect the filtrate in any way & absorb very little
of the substance being filtered
Sterilizing culture media - Filtration
Filters
Membrane filters are suitable for filtering
small volumes of fluid b/c they can be
placed in a Swinnex type filter holder
which can be attached to a syringe

 Swinnex filter holders are available to

take membrane filters of dia. sizes 13
mm, 25 mm & 47 mm

Swinnex holders made from

polypropylene & polycarbonate can be
autoclaved & used many times
Sterilizing culture media - Filtration
Filters

Membrane filters made from cellulose nitrate are also

autoclavable

 They are available in a variety of pore sizes, with 0.22 m

being required for sterile filtration

 The fluid being filtered should be relatively clear to pass

through a 0.22 m porosity filter

 Cloudy fluids should first be passed through a less fine filter

Sterility testing
For routinely media to which blood or other substances
have been added after autoclaving

For ‘sterile’ media in screw-cap tubes or bottles, test for

contamination by incubating 5% of the batch at 35–37 C
o/n

Contamination by Mo’s capable of o/n growth will be shown

by a turbidity in a fluid medium & growth on or in a solid
medium
Sterility testing
Media in petri dishes are examined for contamination
immediately before use

All media, even those that have been sterility tested at the time
of prep, should always be checked visually immediately
before being inoculated for any change in appearance
that could indicate contamination or deterioration

 IMP during the hot season & when the humidity is high
Performance testing
The Central or Regional MB lab should supply district
labs with standardized tested media whenever possible

If not, each lab should set up its own quality control of the
media it prepares

A set of control organisms (stable stock strains) will need to

be obtained from the Regional or Central Public Health
Lab (or from a commercial source) & these organisms
maintained with regular sub-culturing
Performance testing –
Sources of well-characterized stable strains of control bacteria
● National Collection of Type Cultures (NCTC)

● American Type Culture Collection (ATCC)

● Centers for Disease Control (CDC)

● Mast Diagnostics supply QC sticks consisting of lyophilized

gelatin pellets of Mo’s derived from ATCC or NCTC strains

● Oxoid supply QC organisms (ATCC strains) on loops (Culti-

Loops in packs of 5 loops or packs of 100 loops)
Performance testing – Control of NA,
BA & CA
Inoculate slopes or quarter plates of the medium to be tested
with a 5 hour broth culture of each control organism

Use a straight wire to inoculate the medium & a wire loop to

spread the inoculum

Depending on the species, incubate aerobically or in a CO2

enriched atm at 35–37 C

After o/n incubation, examine the cultures for the degree of

growth, size of colonies & other characteristics such as alpha-
or beta-hemolysis
Performance testing – Control of a
differential medium
Inoculate quarter plates of the medium to be tested with a 5 hour
broth culture of each control species

Use a straight wire to inoculate the medium & a wire loop to spread
the inoculum

Incubate aerobically at 35–37 C

After o/n incubation, examine the cultures for the differential

characteristics of the medium

Record the results of each control species & compare with the
results of previous test
Performance testing - Control of a transport medium
 Immerse in the medium a swab of the specimen
containing the pathogen(s) to be preserved
 E.g.:
 Urogenital swab containing N. gonorrhoeae - Amies medium
 Faecal swab containing Shigella or Salmonella - Cary-Blair
medium

 Leave the inoculated transport medium at RT (protected

from direct light) for the length of time the medium is intended
to preserve the viability of the pathogen(s) it contains

 After this time, inoculate the swab on an appropriate

medium to check for viability of the pathogen
Control species
 Most species of bacteria required to control the culture media
used can be maintained in:

 NA deeps covered with sterile mineral oil

 Semisolid NA, on slopes of Dorset egg medium

 Cooked meat medium or Amies transport medium

 Control species of anaerobes are best preserved in cooked

meat medium
Quality control of commonly used culture media
Quality control of commonly used culture
media
Labelling & storage of culture media & additives
Dehydrated culture media & dry ingredients should be stored
at an even temp in a cool dry place away from direct light

Container tops must be tight-fitting & in humid climates,

tape-sealed

Additives (blood, serum, antimicrobials in solid form, urea &

CHO solutions) require storage at 2–8 C

All additives should be allowed to warm to RT before being

used
Labelling & storage of culture media & additives
Antimicrobial solutions : stored frozen at 20 C in the
amounts required

Plates of culture media : stored at 2–8 C in sealed plastic bags

Most media in screw-cap tubes or bottles : stored at RT (20–28 C)

Prepared media should be stored in the dark

When in use, the media must be protected from direct light,

especially sunlight
Sterilizing glasswares in a hot air
oven
 Dry heat, a temp of 160 C held for 45–60 mins, timed from
when the items in the oven have reached this temp

A heating up time (of up to 1 hour) must therefore be allowed

A cooling time is also necessary to enable the items in the oven

to cool slowly

The oven door must not be opened until the temp inside the
oven has fallen to about 50 C
 To avoid cracking the glassware
 To prevent air which may contain contaminating organisms
being drawn into the oven
Sterilizing glasswares in a hot air oven
 A small capacity hot air oven of the convection
type is adequate for most labs

To enable maintenance & any repairs to be

carried out locally, an oven fitted with a
simple hydraulic thermostat and analogue
(dial) thermometer is recommended in
preference to a microprocessor controlled
oven

The oven must be fitted with a protective over-

heat cut out device

The more expensive microprocessor controlled

ovens are usually fitted with a fan, temp chart
recorder, port for thermocouples & a door
Sterilizing glasswares in a hot air oven
Items suitable for sterilizing in a hot air oven (at
160 C) include:

 Glass or aluminium petri dishes (not plastic dishes)

 Glass tubes (rimless) fitted with aluminium caps or with

non-absorbent cotton wool plugs

 Bottles with aluminium caps lined with silicone rubber .

Autoclaving is also suitable for bottles

 Glass flasks & cylinders (cover the open end with

aluminium foil or paper, tied on with string)

 Glass pipettes (graduated & pasteur) with ends

Sterilizing glasswares in a hot air oven
Items suitable for sterilizing in a hot air oven
(at 160 C) include:

 Plugged to a depth of about 20 mm with

nonabsorbent cotton wool

 Nylon or glass syringes (not polypropylene or other

plastic)

 Metal needles, lancets, and forceps (not plastic)

 Dry swabs in tubes, plugged with non-absorbent

cotton wool.
HOW TO DISPENSE CULTURE
MEDIA
Introduction
Media should be dispensed in a clean draught-free room

Most fluid media are dispensed into screw-capped bottles

or tubes & then sterilized by autoclaving

Sterile media must be dispensed into sterile petri dishes,

tubes or bottles using an aseptic technique
Dispensing sterile media into petri
dishes
Lay out the sterile petri dishes on a level surface

Mix the medium gently by rotating the flask or bottle

 Avoid forming air bubbles

Flame sterilize the neck of the flask or bottle & pour 15–20 ml of
medium into each dish (90–100 mm dia.)

 If air bubbles enter while pouring, rapidly flame the surface of the
medium before gelling occurs

Rotate the dish on the surface of the bench to ensure an even layer
of agar
Dispensing sterile media into petri
dishes
When the medium has gelled & cooled, stack the plates &
seal them in plastic bags to prevent loss of moisture & reduce
the risk of contamination

Do not leave the plates exposed to bright light especially

sunlight

Store at 2–8 C

NB:
Agar plates should be of an even depth (not less than 4 mm) &
of a firm gel
Dispensing&solidifyinghighproteinmedia(inspissation)

High protein media are dispensed aseptically in screw-capped

bottles & solidified in a sloped position at a controlled temp
(75–80 C) for 1–2 hours

 E.g. of high protein medium: Dorset egg medium & Loeffler

serum medium

Solidification of protein media using heat to coagulate the

protein = INSPISSATION
Dispensing&solidifyinghighproteinmedia(inspissation)

 Inspissation can carried out in :

 An inspissator (water-jacketed container which allows water
vapour to enter the inspissating area)

 A 75–80 c thermostatically controlled water bath or

oven (over a tray of water to prevent drying of the medium)

The bottle tops should be left loose during inspissation

To prevent bubbles forming in the medium, the temp should be

raised slowly & must not be exceed 80 C