Week 14 MP - Inoculation and 2nd inoculation of Stenotrophomonas maltophilia

Hi everyone, for this week, I am going to post the experiments that will be conducted following culturing of S. maltophilia, which are namely the inoculation and 2nd inculation of S. maltophilia.

After the culturing of S. maltophilia on LB agar, single isolated colonies should be observed on the LB agar after an overnight incubation at 37°C. It should be noted that not all strains of S. maltophilia have simialr growth rate, hence some strains of S. maltophilia may not yield colonies after an overnight incubation. Nevertheless, most strains of S. maltophilia produce colonies after an overnight incubation.

Inoculation of S. maltophilia into LB broth

Principle: To allow the growth and adaptation of S. maltophilia in LB broth at 37°C.

Materials needed:

· 1 sterile 20ml Luria-Bertani (LB) broth (in 50ml tubes)
· Plastic inoculating loops
· S. maltophilia
· NUAIRE Biosafety Cabinet Class II (BSC 2)
· 4°C fridge
· 37°C Orbital shaker incubator (for warming and incubation of LB broth)
· 37°C incubator (for culturing purpose)
· 70% ethanol
· Marker pen
· Scott® C-Fold towels
· Parafilm
· Sticky tapes
· Biohazard bag
· A pair of Sourcelink Powder-Free Latex Medical Examination Gloves (PPE)*
· Clean lab coat (PPE)*
· Covered shoes (PPE)*

*PPE-Personal Protection Equipment

Methods:

1. Incubate the sterile 20ml LB broth into the 37°C Orbital shaker incubator.
2. Swab the work surface of BSC 2 with 70% ethanol.
3. Using an inoculating loop, extract a single isolated colony from the LB agar taken from 37°C incubator.
4. Dip the inoculating loop into the 2oml LB broth and mix it.
5. Discard the inoculating loop into the biohazard bag and label the LB broth.
6. Incubate the LB broth at 37°C in 37°C Orbital shaker incubator overnight while shaking the LB broth with the shaker function of the incubator (Note: The cap of the 50ml centrifuge tube (containing the LB broth and S. maltophilia) must be loosen and tape before placing it into the incubator. This is because S. maltophilia is an obligate aerobe and requires oxygen to grow. By shaking allows air circulation and encourages homogeneous growth of S. maltophilia within the LB broth).
7. Parafilm the LB agar into biohazard bag.
8. Swab the work surface of BSC 2 with 70% ethanol.

Explanation of methods:

Step 1: The LB broth must be warmed up to 37°C so that S. maltophilia does not need to adapt to different temperatures and grow optimally at 37°C. 20ml LB broth is chosen but not any other volume is because it was found that 20ml is the optimal volume for growth of S. maltophilia.

Step 2: This is to ensure the work surface is sterile before any work can proceed. Working inside a BSC 2 is necessary to ensure safety of operator as S. maltophilia is classified as a biosafety class II pathogen. A biosafety class II pathogen is pathogenic and is capable of causing diseases in humans.

Step 3: Self-explanatory

Step 4: Mixing is done is ensure all the S. maltophilia is released into the LB broth and ensure S. maltophilia is evenly distributed in the media.

Step 5: The inoculating loop is considered as biohazardous and should be discarded into biohazard bag (proper disposal). Labeling is done to facilitate identification.

Step 6: This is to allow the growth of S. maltophilia in the LB broth.

Step 7: The LB agar plate (containing the remaining colonies of S. maltophilia) should be parafilmed before disposal. It ensures a safe disposal of bacteria. By sealing the LB agar plate, it prevents oxygen from reaching into the plate and kills all the S. maltophilia.

Step 8: This is to disinfect the work surface.

Result: The LB broth turns cloudy, indicating the growth of S. maltophilia.


2nd inoculation of S. maltophilia into LB broth

Principle: To remove dead cells, debris and toxic metabolic waste products from cells of S.maltophilia and re-inoculate cells into fresh LB broth to prevent overcrowding

Materials needed:

· One 20ml of LB broth (containing S. maltophilia from inoculation)
· Three 20ml of fresh LB broth (for 2nd inoculation, 3 for 1 isolate)
· One 40ml of LB broth (for washing)
· Sterile PBS (phosphate buffered saline) (in 50ml tube)
· 37°C Orbital shaker incubator
· CO8000 Cell density meter
· Centrifuge HERMLE Z 383K machine
· BSC 2
· Centrifuge tube rack
· Disposable pipettes
· Pipettor
· Pipette
· Pipette tips
· 4 cuvettes (1 blank, 3 for each isolate)
· 70% ethanol
· Marker pen
· Scott® C-Fold towels
· Biohazard bag
· Waste bottle
· A pair of Sourcelink Powder-Free Latex Medical Examination Gloves (PPE)*
· Clean lab coat (PPE)*
· Covered shoes (PPE)*


*PPE-Personal Protection Equipment

Methods:

1. Incubate three 20ml LB broth into the 37°C Orbital shaker incubator.
2. Centrifuge the 20ml LB broth (containing the S. maltophilia from inoculation) at 3000xg at 10°C for 20 minutes.
3. Swab the work surface of BSC 2 with 70% ethanol.
4. Decant the supernatant into the waste bottle.
5. Resuspend the cell pellet with 10ml LB broth.
6. Centrifuge at 3000xg at 10°C for 20 minutes.
7. Decant the supernatant into the waste bottle.
8. Resuspend the cell pellet with 10ml LB broth.
9. Centrifuge at 3000xg at 10°C for 20 minutes.
10. Decant the supernatant into the waste bottle.
11. Resuspend the cell pellet with 20ml LB broth.
12. Prepare 4 cuvettes (1 blank and 3 for sample - 1000ul PBS acts as blank and 100ul S. maltophilia + 900ul PBS for each cuvette).
13. Take OD600 readings (3 times and take an average) using CO8000 Cell density meter .
14. Calculate the volume of S. maltophilia required to inoculate into each 20ml LB broth to achieve 5X10^7 cells in each 20ml LB broth.
15. Pipette the calculated volume of S. maltophilia in 20ml LB broth into the each of the 3 fresh 20ml LB broth.
16. Incubate the three 50ml tubes of 20ml LB broth at 37°C in the 37°C Orbital shaker incubator for 16 hours (Note: The cap of the 50ml centrifuge tube (containing the LB broth and S. maltophilia) must be loosen and tape before placing it into the incubator. This is because S. maltophilia is an obligate aerobe and requires oxygen to grow. By shaking allows air circulation and encourages homogeneous growth of S. maltophilia within the LB broth).
17. Discard the remaining LB broth (from inoculation) and wastes into biohazard bag (if full, autoclave the bag).
18. Swab the work surface of BSC 2 with 70% ethanol.

Explanation of methods:

Step 1: The LB broth must be warmed up to 37°C so that S. maltophilia does not need to adapt to different temperatures and grow optimally at 37°C. 20ml LB broth is chosen but not any other volume is because it was found that 20ml is the optimal volume for growth of S. maltophilia.

Step 2: This is to obtain the cell pellet and discard any supernatant (contains dead cells, waste products of cells etc)

Step 3: This is to ensure the work surface is sterile before any work can proceed. Working inside a BSC 2 is necessary to ensure safety of operator as S. maltophilia is classified as a biosafety class II pathogen. A biosafety class II pathogen is pathogenic and is capable of causing diseases in humans.

Step 4: Self-explanatory

Step 5- 10: The cells (S. maltophilia) are washed twice to remove any dead cells, debris and waste products that may affect the growth of S. maltophilia.

Step 11: Since the cells are ultimately inoculated into 20ml LB broth, we should resuspend the cells into similar volume of LB broth before taking of OD600 readings.

Step 12-13: The blank is used for taring of the cell density meter and the cells are diluted 10X before taking OD600 readings.

Step 14: Formula is as follows- (5X10^7)/(Average OD600 reading X 10^10) X 1000

Step 15: The reason for inoculating into three 20ml LB broth is to prevent overcrowding of cells.

Step 16: To allow the growth of S. maltophilia at 37°C .

Step 17: All wastes are considered biohazardous and should be autoclaved.

Step 18: To disinfect the work surface.

Results: The LB broth turns cloudy, indicating the growth of S. maltophilia.

The next experiment will be chloroform shock to extract the periplasmic proteins of S. maltophilia. Please refer to Benjamin's post for more details on chloroform shock.

Alright, till next time...!

Han Yang
TG01
0606190G