Agar plate

An agar plate is a sterile petri dish that contains agar plus nutrients, and is used to culture bacteria and fungi.

Table of contents

1 Preparation of Agar Plates
2 Inoculation techniques

2.1 Streaking
2.2 Christmas tree
2.3 Stab culture
2.4 Preparing a lawn

3 Incubation of agar plates
4 Types of agar plates
5 Safe Disposal of Agar plates

Preparation of Agar Plates

Most types of agar are purchased in powder form. They are dissolved in distilled water as per their instructions. It usually necessary to gently boil the mixture to facillitate dissolving, this can be done in a microwave, or over a gently flame. Once dissolved the agar needs to be sterilised by pouring it into a conical flask, then sealing the top with a cotton wool wad, and finally covering the cotton wool with a loose layer of aluminium foil. This is then autoclaved for 15 minutes. The sterile agar is then allowed to cool to 50°C. This is just above the setting point, Pouring at this temperature helps prevent condensation forming on the lid.

Before the plates are poured, every care is taken not to contaminate them with stray bacteria.

Hands are washed with soap and hot water.
The bench is wiped with ethanol or some other disinfectant
A bunsen burner is set up with a gentle blue flame. This will be used to sterilise the mouth of the flask, and also provides a reasonably sterile environment in the vicinity.
The number of plates to be poured are placed on the bench, with their lids still on.
The aluminium is removed and discarded. The cotton wool is removed with the little finger. It is held in the little finger the whole time, not put down.
The mouth of the flask is flamed to kill bacteria on the outside of the rim.
The lid of the plate is lifted just high enough to allow the plate to be poured, and is quickly half filled with agar.
The lid is replaced, the plate swirled gently and left where it is on the bench for at least 20 minutes to solidify.
Once all the plates are poured, the flask mouth is reflames and the cotton wool reinserted. Any unused agar is still sterile

Inoculation techniques

Streaking

The most common method of innoculating an agar plate is streaking.

Streak plates

With this method, a small amount of sample is placed on the side of the agar plate (either with a swab, or a drop from an inoculating loop if it's a liquid).
A sterile loop (flamed until red hot, then cooled by touching the center of the agar) is then used to spread the bacteria out one direction from this. This is done by moving the loop from side to side, entering the initial site of inoculation.
The loop is then sterilised (by flaming) again and the first streaks are then spread out themselves.
This is repeated 2-3 times, moving around the agar plate.

What should happen is that single bacteria get isolated by the streaking, and when the plate is incubated, the resulting colony will have started from just one bacterium.

This is used for culture of urine. A small loop is dipped in the urine, and a single streak is made down the middle of the agar plate. Then the loop is swayed in and out going at through the streak multiple times at right-angles to the first streak.

Stab culture

A needle is flamed then immersed in the culture. It is then stabbed into a small sterile jar of nutrient agar. If the bacteria are anaerobic they grow, otherwise they do not.

Preparing a lawn

A lawn is often used for antibiotic sensitivity testing. What is needed is an even and complete spread of growth all over the agar plate. Around an antibiotic disc there will be an area of no growth, the diameter of this can be measured to find out whether that bacterial strain is resistant to the antibiotic.

One way to prepare a lawn, is to use a 0.5 McFarland suspension of bacteria in saline. (This means the saline is made just slightly turbid.) A sterile swab is dipped into this, then it is moved side to side down the whole agar plate so all the area is covered. The plate is rotated 90° and the swab moved side to side perpedicularly to the first time. This is down once more with the swab rotated 45°.

Once a lawn has been prepared, a small disk of filter paper is soaked in antibiotic and placed on the plate. After incubation there will be a ring of zero growth visible around the filter paper if the lawn bacteria are sensitive to that antibiotic. A collection of small disks each soaked in a different antibiotic, and attatched to a larger ring can be purchased commercially. They are known as Mast rings and can be used to test the sensitivity of an organism to a range of antibiotics all at once.

Incubation of agar plates

Most plates are incubated at 37°C in 5% CO₂, which is the temperature and conditions that most of the body's bacteria will grow. Special incubators can maintain these conditions.

Some bacteria must be incubated anaerobically (without any oxygen). These can be placed in containers, along with a substance that removes oxygen, and the tightly sealed container placed in the regular incubator.

Fungi, and some bacteria (e.g. Yersinia sp.) should be incubated slightly cooler. This is usually 30°C, and room air often is used.

Yoghurt bacteria grow at much higher temperatures. (typically ~45°C) They are therefore particulally safe to use when teaching microbiology, especially to children.

Campylobacter is a difficult bacteria to grow. It needs special agar plates, plus its own microaerophilic environment.

Types of agar plates

Blood agar - contains blood cells from an animal (e.g. a sheep). It will grow most bacteria.
Chocolate agar - the contains lysed blood cells, and is used for growing fastidious (fussy) respiratory bacteria.
Neomycin agar - contains the antibiotic neomycin.
Sabouraud agar - used for fungi. It contains gentamicin and has a low pH that will kill most bacteria.
Thayer-Martin agar - chocolate agar designed to isolate Neisseria gonorrhoeae.
XLD agar - Xylose Lysine Deoxycholate agar. It is used for the culture of stool samples, and contains two indicators. It is formulated to inhibit gram-positive bacteria. The growth of gram-negative bacilli is encouraged. The colonies of lactose fermenters appear yellow.
Nutrient agar, safe to use in school science laboratories because it does not selectively grow pathogenic bacteria.

Safe Disposal of Agar plates

Plates, onced finished with must be made safe before throwing away. The usual method is to place inside an autoclave bag and then autoclaved at 121°C, 103 KPa (15 psi) for 15 minutes. The plates will melt (hence the bag). After about 20 minutes the autoclave will have cooled down. The bag can then safely be thrown away. If no autoclave is available an ordinary domestic pressure cooker can be used, or, in a hospital or professional lab an incinerator may be used.

Other equipment should be placed in a suitable disinfectant such as Virkon for 24 hours.