Jan 23, 2023

Public workspacePreparing Biolog Growth Plates V.1

This protocol is a draft, published without a DOI.
  • 1[North Carolina State University]
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Protocol Citation: Carlos Carlos Goller, Carly Sjogren 2023. Preparing Biolog Growth Plates. protocols.io https://protocols.io/view/preparing-biolog-growth-plates-cht6t6re
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: In development
We are still developing and optimizing this protocol
Created: October 12, 2022
Last Modified: February 28, 2024
Protocol Integer ID: 71262
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 Tryptic Soy Agar 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
Materials
Activity 1:
  • One 200 μl multichannel micropipette (p200)
  • Micropipette tips for p200
  • One 96-well plate
  • Chem wipes
  • One container with water with food coloring (yellow)
  • One container with water with food coloring (green)
  • Tip disposal container

Activity 2:
  • 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
  • Chem wipes
  • 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
Set your p200 multichannel pipet to Amount100 µL

Pipetting
Load 8 tips onto each gasket of the multichannel pipet.
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 image or the descriptions below)
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.
If using colors other than yellow and green, make your own pattern.

Reference image:

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.
Critical
Have each researcher do two to three columns of the plate to practice.

Activity 2- Innoculation
Activity 2- Innoculation
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 Turbidimeter with bacterial sample in the inoculation fluid in a tube
Collect bacteria from your streak plate by touching the Inoculatorz™ swab to the bacteria
Safety information
Be gentle: do not push the swab through the agar media

Open your 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 plates preparation
Activity 3-Biolog plates preparation

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.
Check that all wells have liquid
Cover the GEN3 microplate with its lid and eject the pipette tips.
Incubate at Temperature28 °C for . Duration72:00:00 - Duration120:00:00 hours

Reference image:



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?

Incubation
Overnight