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

Record the results of each control species & compare with the results of previous
performance tests
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 interlock
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

The surface of the medium should be smooth & free from bubbles
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