Friday, September 26, 2008

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

14 comments:

Fluid collectors said...

HI hanyang!
What do u mean by homogeneous growth?

And which is the better way to grow S. maltophilia? broth or agar?
Thanks!
Shihui
0607135A

Fluid collectors said...

Hello.

How do you ensure that 'dead cells, debris and toxic metabolic waste products from cells of S.maltophilia' are not present after 2nd inoculation of the organism into the broth?

And after 2nd inoculation into the broth, do you have to subculture the broth onto any agar?

Thanks!

-Li Ping-
0607498C
TG o2

SIP said...

Hi Hanyang,

I'm just curious why is LB agar chosen instead of MacConkey or the others?Is it because S.maltophilia is a non-lactose fermenter? And for the waste bottle, do u use sodium hypochloride or virkon solution and what is the concentration of the solution u use and why?

Thanks
Ying Chee
TG01

De Incredibles said...

Hi there
How u noe "20ml is the optimal volume for growth of S. maltophilia"....do u do optimisation? like try out with different volume of LB broth?

Lim Xin Ni
TG02

tg01 group 2 said...

Hi,

i jus wanna know what is a Orbital shaker incubator? what is its function? i dun mind knowing the principle of the equipment/machine...

From Ivan

group1 said...

Hi hanyang!

is there a chance of contamination? if yes, how do you know if it's contaminated?

i notice that you use cell density meter to check the concentration of the cells. does cell densiy meter works like spectrophotometer?

u mention "not all strains of S. maltophilia have simialr growth rate" what do u mean by other strains?

Thanks!
Yu Mei
TG01

SIP said...

hey hanyang!

Can i know where you get the source of S.maltophilia? (eg.urine, sttool, swab)

And you mentioned that if LB broth turns cloudy, it indicates the growth of S. maltophilia... Can it indicates other possiblities? (eg like what yumei mentioned contamination?)

Thanks!

Cheers,
Huimin
Tg01 =)

tg01 group 2 said...

Hi

What are the methods of detecting Stenotrophomonas maltophilia?

Because you simply assumed when the LB broth turns cloudy, it indicates the growth of S. maltophilia.

Do you do a gram stain? Like to find out if it is gram negative?

Thank you
Ernest
0606330i

tg01 group 2 said...

Wah, that was fast...! 8 comments received within a day... sweat...!

Hi Shihui,

Homogeneous growth means equal or uniform growth.

Eh, We must first culture the S. maltophilia on LB agar, get the isolated colonies before inoculating them into the LB broth. Culturing is first step and inoculation and 2nd inoculation are second and third step of extraction of periplasmic proteins of S. maltophilia respectively. If you want to ask me which is the better way, I would say it is the conventional method which is streaking on LB agar. I feel that any bacteria should first be allowed to grow and multiply on suitable agar before they are used for any application. I have never tried directly inoculating the fresh S. maltophilia stock into the LB broth directly. Chances are the bacterium won't grow.

Hi Li Ping,

The washing step is done TWICE so as to ensure most of the dead cells, debris and toxic metabolic waste products are removed from the cells. Make sure that all cell pellet are resuspended in LB broth and centrifuge at the condition I have mentioned. It should do the trick!

Eh, we do not have to subculture the broth onto any agar. Following 2nd inoculation, we will perform chloroform shock which is mentioned by Benjamin's post in the earlier weeks. Please refer to his post on more details on how to extract the periplasmic proteins from S. maltophilia.

Hi Ying Chee,

LB agar is generally suitable for the growth of gram-negative bacteria such as S. maltophilia. It is rich in nutrients (e.g. peptides, vitamins, trace elements and minerals) that will provide optimum growth for the bacterium. Yes, S. maltophilia is a non-lactose fermenter.

We do not use sodium hypochloride or virkon solution. We simply autoclave the waste.

Hi Xin Ni,

For us, we do not do optimisation as to whether 20ml of LB broth is the optimum volume for inoculation. Perhaps for previous batches of MP students or through research, they have found out that 20ml LB broth is sufficient and good enough to support the growth of S. maltophilia.

Hi Ivan,

Orbital is the brand of the machine. LOL...! As the name implies, it is a 2-in-1 incubator which has a shaker function as well as allowing incubation at desired temperatures. It is just as simple as swtiching on the incubator and adjust the speed of shaking motion (rpm)and start incubating the LB broth...! The shaker function can be turned off. Heat is generated from the fan/motor and provide the temperature you need. When we are incubating the LB broth, it is good to tape the cap of the tube as we have loosened it to prevent spiilage.

Hi Yu Mei,

The chance of contamination is very, very slim as we are performing the experiment within the BSC 2. Since we abide to strict aseptic techniques, there has not been any contamination thus far. Strict aseptic techniques should always be followed when doing microbiology work. One precaution you could take is that if you feel that the inoculating loop has touched the work surface of BSC 2 which is non-sterile, do not dip it into the LB broth. This will definitely introduce contamination into the LB broth, thus growing unwanted bacteria.

The cell density meter works similarly as a spectrophotometer but just at different wavelength from the spectrophotometer(595nm -for Bradford assay), which is at 600nm. The results obtained from the cell density meter is comparable to spectrophotometer and it is very convenient to use it.

We are culturing 11 strains of S. maltophilia, of which some are clinical isolates while some are environmental isolates. The environmental isolates would take a longer time to adapt to 37 degree celcius (be it growing in LB broth or LB agar) as it is obtained from the environment where the temperature is at around 28 degree celcius. Thus, these environmental isolates tend to grow slower than the clinical isolates which are obtained from patient specimens at 37 degree celcius.

Hi Huimin,

The S. maltophilia are obtained from ATCC (American Type Culture Collection) and from patient samples such as cerebrospinal fluid and blood culture. The S. maltophilia isolated from patient samples are processed by ATCC and we just have to purchase from them.

During Basic Microbiology, we have learnt that cloudy media indicates growth of bacteria. In order to ensure you are inoculating just S. maltophilia but not any other bacteria into the LB broth, you have to ensure your culturing step is done properly (e.g. follow strict aseptic techniques). This will assure you that what you grow on the LB agar plate are purely S. maltophilia but not any other bacteria and when during inoculation, you will be assured that what you pick up from the LB agar and inoculate into LB broth is S. maltophilia only. Thus, the cloudy LB broth can only indicate the growth of S. maltophilia but not any other bacteria. In addition, the LB broth is sterile as it is autoclaved before use. Hence, this provides more assurance that the LB broth is close to 99% free of any organism and pure.

Hi Ernest,

You can refer to my reply to Yumei and Huimin for the reason I am assured that the cloudy LB broth indicates the growth of S. maltophilia.

Since each strain of S. maltophilia is contained in individual vials and they are obtained from a trustworthy source such as the ATCC, you can be rest assured that what you pick up from the vial and culture on the LB agar is purely the strain of S. maltophilia you want and what you pick up (the colonies) from the LB agar and inoculate into the LB broth is 100% S. maltophilia UNLESS aseptic techniques are not followed.

We do not perform any gram-staining but gram-staining is one of the method to detect S. maltophilia. The purpose of our project is not so much on identification but more on protein work.

I hope my reply to everyone is satisfactory...! Phew...! =)

Han Yang
TG01

SIP said...

Hi hanyang

Why is there different growth rate for S.maltophilia?? Isn't all the growth rate for bacteria the same??

Justina
TG01

De Incredibles said...

Hi Hanyang,
just to confirm..we can also warm the LB broth in the 37 degree celcius water bath ya? I thought it would be must faster than the incubator. hmmz, so no special reasons for warming in incubator rite?

Jean Leong
TG02

tg01 group 2 said...

Hi Ivan,

After further clarification, orbital is NOT the brand of the shaker incubator but is the way how the shaker shakes. Orbital means revolving shaking motion. The brand of the shaker incubator is MRC.

My apology...

Han Yang
TG01

tg01 group 2 said...

Hi Jean,

Haha, you think too much le...Warming the LB broth in either incubator or water bath is perfectly alright and there are no special reasons...

Han Yang
TG01

tg01 group 2 said...

Hi Justina,

Different strains of S. maltophilia have different growth rate and this is most likely due to the different environments the bacteria are isolated from. For example, if the bacteria is isolated from human body, it would adapt to 37 degree celcius much faster and better and have a faster higher growth rate. If the bacterium is isolated from the environment, naturally the bacterial will be used to the environmental temperature and thus if you grow the bacteria at 37 degree celcius, it would take a much longer time to adapt to 37 degree celcius and have a slower growth rate.

Hope you can understand!

Han Yang
TG01