Jun 10, 2022
Making Blood Agar Plates
- 1University of Calgary
Protocol Citation: Laura Sycuro, Ramon Cortez, Shae Konschuh 2022. Making Blood Agar Plates. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj6p7xlk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 06, 2022
Last Modified: June 10, 2022
Protocol Integer ID: 62139
Keywords: Blood agar plates
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Abstract
Purpose
Blood agar is agar base enriched with 5% blood. It is an enriched medium often used to grow fastidious organisms and to differentiate bacteria based on their hemolytic properties.
Key concepts
The agar base can vary – Brucella is what is generally prefered for the growth of diverse vaginal organisms, but Casman and Columbia have also been used and shown to support a broad variety of organisms.
Materials
Supplies
- Agar base (can be media base already containing agar, or liquid media base to which you add the agar separately)
- Distilled water
- Glass media bottles
- Blood (we usually use defibrinated sheep blood that is less than 1 month old)
- Sterile Petri dishes
- 25 mL serological pipet
- Serological pipettor
Equipments
- Autoclave
- Water bath
- Bunsen burner
- Stir plate
Safety warnings
Wear a regular (fabric) lab coat when you pour plates. This helps ensure your sleeves/clothing won't become soiled with blood and won't come into contact with the Bunsen burner flame. Tie long hair back.
Preparation
Preparation
Mix appropriate amount of agar base into distilled water in a glass media bottle.
- 500 mL agar in a 1 L bottle makes ~25 plates.
- Each bottle of powdered media lists the grams of powdered media needed for 1 L of media. Use half this amount for 500 mL.
Stir media with stir bar until completely dissolved.
Note
Leave the stir bar in the bottle during autoclaving.
Autoclave
- For 1 or 2 bottles of Brucella agar media, autoclave for 15 minutes.
- For 3 or more bottles of Brucella agar media, autoclave for 30 minutes.
- For Columbia agar media, autoclave for 45 minutes.
Note
**Be sure to label the bottle and the autoclavable pan with 'Agar' and requested autoclave time ('15 minutes'). Also can be helpful if you add ~1 inch of water to the bottom of the pan (they won't always do this for you).
After dropping off downstairs for autoclaving, turn water bath on and set to 55 °C . Remove blood from refrigerator and allow to warm on the bench.
- Check and make sure no one else needs water bath at lower temperature for the next 1-2 hours.
- Use the freshest blood available and generally do not use blood that is over 1 month old.
Pouring
Pouring
30m
30m
Pick up media from downstairs shortly after run completes (they should place it in a 55 °C incubator downstairs after the run).
Note
**Remember to bring thermal gloves with you when you go downstairs to retrieve your media from the incubator. It may still be hot!!
- Place in 55 °C water bath for 20-30 minutes.
- Media is ready to pour when bottle is warm, but not hot to the touch.
Mix blood (at room temperature) by gently swirling (rubbing the bottle between your hands works well).
Prepare plates and pouring area.
- Clear and clean area of bench near Bunsen burner.
- Arrange sterile plates right side up in small stacks of 4-5.
- Stripe plates if desired.
Bring blood, a 25 mL serological pipet, and the serological pipettor to the media making area (stir plate).
Remove 1 bottle of media from water bath and place on stir plate.
While media is being stirred, pipet 25 mL of blood into 500 mL of media (final concentration of5 % (v/v) )
Stir for 1 minute or until mixed.
Move to pouring area and pour plates immediately.
- Flame lip of bottle at the start of each stack of plates.
- When bottle is empty, carefully flame plates to remove bubbles.
Gently push plates to back of bench (away from Bunsen burner hose) to solidify and then clean up bench and rinse the media bottle.
- All papers and pipets with blood on them get disposed in the yellow biohazard waste bin (near fume hood).
Prepare to pour second batch of plates if needed.
Completely finish with one batch of plates before removing a second bottle from water bath.
- Do not add blood to second bottle and then put back into water bath – carry out the pouring procedure from start to finish 1 bottle at a time.