Jan 22, 2025

Public workspacePreparing Biolog Growth Plates V.3

DOI
dx.doi.org/10.17504/protocols.io.e6nvwj5ndlmk/v3
  • 1Biotechnology Teaching Program (BIT), North Carolina State University
  • BIT-Protocols
    Tech. support phone: +91 95134-135 email: ccgoller@ncsu.edu
Nidhi Grover
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwj5ndlmk/v3
Protocol Citation: Carlos Carlos Goller, Carly Sjogren 2025. Preparing Biolog Growth Plates. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwj5ndlmk/v3Version created by Carlos Carlos Goller
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: February 14, 2023
Last Modified: January 22, 2025
Protocol Integer ID: 118848
Keywords: Biolog, Growth, multichannel
Funders Acknowledgements:
Biotechnology Program
Abstract
Overview and Goals
Your bacterial isolate has been grown on agar plates. You practiced pipetting. Now, let’s learn about how your organism grows! What nutrients help your bacterium grow? We will use plates with ninety-six wells with different conditions to examine the growth and metabolic capacity of your isolate. For this, we will use special Biolog plates and your Blood Universal Growth (BUG) media containing plates with your organism. Biolog plates allow researchers to test and visually (colorimetrically!) analyze the growth of bacteria in numerous conditions… up to 30 conditions in one plate! Biolog plates work by having powder chemicals in the wells along with an inoculating fluid and a dye that changes color when an organism is growing (“metabolic dye”).

After completing this lab you will gain the following lab skills:

  • Lab safety and proper personal protective equipment (PPE)
  • Proper use of Biolog plates and multichannel pipettors
  • Proper use of a turbidimeter to adjust bacterial densities
  • Analysis of growth data of bacteria in 96-well plates
Image Attribution
Image created with BioRender.com
Guidelines
Work carefully and keep the tube capped. If necessary, use another swab if you think it has touched another surface.

Do not leave the liquid uncapped longer than necessary to prevent contamination. The tubes are glass: work carefully.
Materials
Activity 1:
  • One 200 μl multichannel micropipette (p200)
  • Micropipette tips for p200
  • One 96-well plate
  • Kimwipes
  • One container with water with food coloring (yellow)
  • One container with water with food coloring (green)
  • Tip disposal container

Activities 2-3:
  • One Inoculatorz™ swab
  • IF-A inoculating fluid
  • Tryptic Soy Agar (TSA) plate with bacteria
  • Biolog GEN3 plate
  • One 200 μl multichannel micropipette (p200)
  • Micropipette tips for p200
  • Kimwipes
  • Tip disposal container
Before start
Review the figures below to learn about bacterial cell structure, shape, and growth.



Bacterial structures:
  • Pilus
  • Flagellum
  • Capsule
  • Cell wall
  • Plasma membrane
  • Nucleoid
  • Ribosome
  • Plasmid





Bacterial shapes:
  • Cocci
  • Coccus
  • Diplococci
  • Diplococci (encapsulated)
  • Staphylococci
  • Streptococci
  • Sarcina coccus
  • Tetrad coccus
  • Bacilli
  • Coccobacillus
  • Bacillus
  • Diplobacilli
  • Streptobacilli
  • Palisades
  • Appendaged bacteria
  • Hypha
  • Stalk
  • Others
  • Club rod
  • Vibrio
  • Spirillum
  • Helical Form
  • Spirochete
  • Filamentous




Bacterial growth phases over time:
  • Lag phase (slow growth)
  • Log phase (exponential growth)
  • Stationary phase (growth plateau)
  • Death phase (decline, depends on the microbe and growth conditions)


Next, read pages 2-5 of this document about the Biolog GEN III MicroPlate™ (text, ~10 min). We will begin by practicing the use of a multi-channel pipette with containers (“reservoirs”) with water and food coloring.
Activity 1: Multichannel pipetting
Activity 1: Multichannel pipetting
24m
24m
Set your p200 multichannel pipet to Amount100 µL

1m
Pipetting
Load eight (8) tips onto each gasket of the multichannel pipet.
1m
Press the plunger TO the first stop.
Critical
Submerge all 8 tips into the water with food coloring.
With thumb control, release the plunger to take up liquid. Check each tip to make sure each has the same volume of liquid.
Note
Note the height of the liquid in the tips. Is it consistent?

Critical
Align each tip with one well across one column of the 96-well plate (refer to the image or the descriptions below)
1m
Yellow water should go in odd numbered columns 1, 3, 5, 7, 9, 11.
Green water should go in the even numbered columns 2, 4, 6, 8, 10, 12.
5m
If using colors other than yellow and green, make your own pattern.

Reference image:
Each researcher will use multi-channel pipette to transfer 100uL of colored solutions to each numbered columns of the 96-well plate. Check that you are delivering the appropriate volume to all wells.
Press the plunger THROUGH the first stop, raise your tips up out of the plate, and then you may release your plunger with thumb control.
1m
Critical
Have each researcher do two to three columns of the plate to practice.

15m
Activity 2: Turbidimiter
Activity 2: Turbidimiter
We will swab bacterial cells from your agar plates (BUG Agar: BUG (Biolog Universal Growth) Agar w/ 5% sheep blood), dilute in Biolog IF-A inoculation fluid, and check turbidity with a turbidimeter.

Calibrate the turbidimeter to a "blank" sample of inoculation fluid in a tube
Using the Biolog Turbidimeter, blank the turbidimeter with a clean tube containing uninoculated IF-A.
Note
Wipe the tube clean of dirt and fingerprints
Because the tubes used are not optically uniform, they should be blanked individually.

Set the 100% transmittance adjustment knob so that the meter reads 100%.
Record reading.
Use a turbidimeter with bacterial sample in the inoculation fluid in a tube
Collect bacteria from your streak plate by touching the Biolog Inoculatorz™ swab to the bacteria
Safety information
Be gentle: do not push the swab through the agar media

Open your Biolog Inoculating Fluid tube (IF) tube and submerge the swab into the liquid. Swirl the swab around in the fluid for Duration00:00:15 to ensure inoculation.
Safety information
Do not leave the liquid uncapped longer than necessary to prevent contamination. The tubes are glass: work carefully.


15s
Cap the tube and gently invert the three (3) times to ensure thorough mixing.
The target cell density is 95%T for our protocol.
  1. Add more bacteria with the swab if necessary to get as close to this value as possible by continuing to add more bacteria with the swab to the tube.
  2. Work carefully and keep the tube capped. If necessary, use another swab if you think it has touched another surface.
Once you've reached the target cell density at 95%T, cap the tube and proceed to the next Activity.
Activity 3: Biolog plate preparation
Activity 3: Biolog plate preparation
2d
2d
Each researcher will use a multi-channel pipette to transfer 100uL of inoculating fluid and bacteria to each numbered column of the 96-well plate. Check that you are delivering the appropriate volume to all wells.
Pour the cell suspension into the multichannel pipette reservoir.
Load 8 tips onto each gasket of the multichannel pipet and fill the tips by drawing up Amount100 µL of cell suspension from the reservoir.

Pipetting
Use the multichannel to add Amount100 µL of diluted bacterial culture to each of the 96 wells of the GEN III MicroPlateTM.
Note
Use the technique practiced in Activity 1.

Safety information
If the tips touch the wells, replace your tips before delivering inoculation fluid to the next column. Work carefully to avoid contamination!


Critical
Eject the pipettor tips if any one tip touches the wells of the plate as this can cross contamination wells with chemicals. Reload tips as needed.
Critical
Check that all wells have liquid
Cover the GEN3 microplate with its lid and eject the pipette tips.
Incubate at Temperature33 °C for Duration24:00:00 - Duration24:00:00 2d hours. For some plates, we will incubate in a heated Agilent/Biotek LogPhase 600 plate reader and take 590 nm/600 nm readings every 15 minutes for 2 days to learn about the growth patterns.

Reference image:

Illustration of Biolog GEN3 plate with several purple wells. Right: icon of calendar with text "Incubate the plate at 33'C for 1 to 2 days"
Clean Up Instructions
  1. Biolog growth plates must be given to an instructor to place in our plate readers to measure viability and growth!
  2. All liquids from Activity 1 (materials with no bacterial contamination) can be poured down the sink and plastic can go in the regular trash.
  3. All liquids containing bacteria must be poured into a beaker for bleach decontamination (can be found on the blue bench paper by this sink labeled "liquid").
  4. Glass tubes can be placed into a beaker (can be found on the blue bench paper by this sink labeled "glass").
  5. All materials from Activity 2 that contact bacteria need to go into the biohazard trash.
  6. All bacterial growth plates can be thrown away in the biohazard trash
  7. Return all reusable materials to our storage drawers.
  8. Wipe down all surfaces with Conflikt and then ethanol.
  9. Remove PPE.
  10. Wash hands.

Note
Critical Thinking Questions for Preparing Biolog Growth Plates

  1. What are some advantages of using a multichannel pipette?
  2. What are some disadvantages of using a multichannel pipette?
  3. During the inoculation, describe how you ensure that you isolate a "pure culture." Why is a "pure culture" needed for this assay?
  4. What is a turbidimeter, and why would measuring turbidity be important for this assay?

2d
Incubation
Overnight
Protocol references
This protocol has been adapted from the Biolog GEN3 Protocol.
https://www.biolog.com/products/microbial-identification-microplates/gen-iii-test-panel/
Acknowledgements
We appreciate the support from the NC State Biotechnology Program and students who have helped improve this and other protocols.