TITLE: Preparing Biolog Growth Plates
AUTHORS: Carlos Carlos Goller, Carly Sjogren

[DESCRIPTION]
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

[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.

[STEPS]
SECTION: Activity 1-Multichannel pipetting
1. Set your p200 multichannel pipet to 100 µL 

SECTION: Activity 1-Multichannel pipetting
2. Load 8 tips onto each gasket of the multichannel pipet.
SECTION: Activity 1-Multichannel pipetting
3. Press the plunger to the first stop.
SECTION: Activity 1-Multichannel pipetting
4. Submerge all 8 tips into the water with food coloring.
SECTION: Activity 1-Multichannel pipetting
5. With thumb control, release the plunger to take up liquid. Check each tip to make sure each has the same volume of liquid. 
 

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

Reference image:
 
SECTION: Activity 1-Multichannel pipetting
7. 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.
SECTION: Activity 1-Multichannel pipetting
8. Have each researcher do two to three columns of the plate to practice.

SECTION: Activity 2- Innoculation
9. Calibrate the turbidimeter to a "blank" sample of inoculation fluid in a tube
SECTION: Activity 2- Innoculation
9.1. Using the Biolog Turbidimeter, blank the turbidimeter with a clean tube  containing uninoculated IF-A.
 

SECTION: Activity 2- Innoculation
9.2. Set the 100% transmittance adjustment knob so that the meter reads 100%.
SECTION: Activity 2- Innoculation
9.3. Record reading.
SECTION: Activity 2- Innoculation
10. Use Turbidimeter with bacterial sample in the inoculation fluid in a tube 
SECTION: Activity 2- Innoculation
10.1. Collect bacteria from your streak plate by touching the Inoculatorz™ swab to the bacteria
 

SECTION: Activity 2- Innoculation
10.2. Open your Inoculating Fluid tube (IF) tube and submerge the swab into the liquid. Swirl the swab around in the fluid for 15 s to ensure inoculation. 
 


SECTION: Activity 2- Innoculation
10.3. Cap the tube and gently invert the three (3) times to ensure thorough mixing.
SECTION: Activity 2- Innoculation
10.4. The target cell density is 95%T for our protocol.
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.
Work carefully and keep the tube capped. If necessary, use another swab if you think it has touched another surface.
SECTION: Activity 2- Innoculation
11. Once you've reached the target cell density at 95%T, cap the tube and proceed to the next Activity.
SECTION: Activity 3-Biolog plates preparation
12.  
Pour the cell suspension into the multichannel pipette reservoir.  
SECTION: Activity 3-Biolog plates preparation
13. Load 8 tips onto each gasket of the multichannel pipet and fill the tips by drawing up 100 µL  of cell suspension from the reservoir.  

SECTION: Activity 3-Biolog plates preparation
14. Use the multichannel to add 100 µL  of diluted bacterial culture to each of the 96 wells of the GEN III MicroPlateTM. 
 

 


SECTION: Activity 3-Biolog plates preparation
15. 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.
SECTION: Activity 3-Biolog plates preparation
16. Check that all wells have liquid
SECTION: Activity 3-Biolog plates preparation
17. Cover the GEN3 microplate with its lid and eject the pipette tips. 
SECTION: Activity 3-Biolog plates preparation
18. Incubate at 28 °C  for . 4320 min - 7200 min  hours

Reference image: