Sunday, August 24, 2008

Week 9 MP - Culturing of Stenotrophomonas maltophilia

Hi everyone...! I have changed my MP due to some unforseen circumstances (which I shall not elaborate further) and it will be somehow similar to Benjamin's MP.

MP title: Development of 2D-Zymogram protocols to detect and identify Stenotrophomonas maltophilia periplasmic proteases and compare their activity at 37°C vs 28°C.

Quick facts on Stenotrophomonas maltophilia (S.maltophilia)
  • Previously known as Pseudomonas maltophilia or Xanthomonas maltophilia
  • Sole member of the genus, Stenotrophomonas
  • Gram-negative bacillus (rod-shaped) which is gram-stained pink
  • Aerobic (require oxygen to grow and survive)
  • Non-fermentative
  • Motile by means of flagella
  • Found widely in environment and hospitals
  • Infects immunocompromised individuals especially

Why study S.maltophilia ?

  • Important nosocomial pathogen
  • Highly antibiotics resistant
  • Little is known of its virulence factors, genetic structure and pathogenicity
  • Ageing population
  • Increased use of surgical equipments

Culturing of S.maltophilia

Principle: To allow the growth of S.maltophilia on LB agar plate and to obtain single, isolated colonies of the bacterium.

Materials

  • S.maltophilia isolates (in vials, frozen state)
  • Disposable inoculating loops
  • LB agar
  • 37°C incubator
  • Biosafety cabinet 2 (BSC 2)
  • 70% ethanol
  • Marker pen
  • Biohazard bag
  • Appropriate personal protection equipments (e.g. Lab coat, covered shoes and gloves)

Methods

  1. Set up BSC 2.
  2. Dry LB agar in 37°C incubator.
  3. Swab vials (containing S.maltophilia isolates), inoculating loops and LB agar with 70% ethanol.
  4. Place the necessary materials into the BSC 2 and arrange them orderly.
  5. Label the LB agar plate.
  6. Streak on the LB agar plate with an inoculating loop (with S.maltophilia).
  7. Dispose used inoculating loops into biohazard bag.
  8. Swab the LB agar plate with 70% ethanol before incubating at 37°C overnight.
  9. Observe for single, isolated colonies.

Explanation of methods

1) To provide a sterile environment for culturing to prevent any contamination. To protect the user from S.maltophilia (safety reason).

2) To prevent the formation of water droplets from condensation as the LB agar are stored in the fridge.

3) Aseptic techniques

4) To facilitate workflow within the BSC 2.

5) To facilitate identification of S.maltophilia isolate.

6) To transfer S.maltophilia onto the LB agar plate.

7) Used inoculating loops are considered biohazard wastes and should be disposed in biohazard bag and autoclave later.

8) To disinfect the outer surface of LB agar plate with 70% ethanol before incubation. Incubation at 37°C overnight allows growth of S.maltophilia optimally.

9) This shows that streaking was done properly (recall streaking techniques from Basic Microbiology) and the colonies will be used for subsequent experiment such as inoculation.

That's all for this week!

Tan Han Yang

0606190G

TG01

23 comments:

hellomedtech said...

hello..

why do you use LB plate?

can other plates be used to isolate the colonies of the bacteria mentioned?

sutiana

tg01 group 2 said...

Hi Sutiana,

Thankz for reading my post!

LB (Luria Betani) agar is a nutrient agar that is generally used for the cultivation of microorganisms (particularly not fastidious microorganisms). It contains peptone (amino acid), yeast extracts and sodium chloride which are formulated to support the growth of microorganisms. LB agar is suitable for student usage and it does not pose any health hazards to user. Since it has been shown that LB agar can be used to culture E.coli, which is a gram-negative bacterium, it can be used to support Stenotrophomonas maltophilia too as it is also a gram-negative bacterium.

In addition, the preparation of LB agar is simple.

The bottom line is that LB agar is able to meet the nutritional requirements of the bacterium I am culturing and support its growth.

Han Yang
TG01

De Incredibles said...

Hi han yang,

Can u explain a little on what is 2D-Zymogram? And also, is there a reason why u wana compare their activity at 37°C vs 28°C? As in why these two speicific temperature?

Thanks alot!

Zhenling =)
TG02

tg01 group 2 said...

Hi Zhenling,

Thankz for reading my post!

2D-zymography is a two-step electrophoretic technique used to separate proteins based on 2 dimension separation steps. The first dimension step is known as isoelectric focusing (ISF) while the second dimension step is known as SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).

The first dimension step separates proteins based on isoelectric point [pI](the specific pH at which the net electrical charge of proteins is zero), a characteristic of protein. The second dimension step separates proteins based on molecular weight. SDS is an anionic detergent that denatures proteins and linearise them. SDS bind to proteins at 1.4g SDS/g protein and give proteins a constant mass per unit charge. With the addition of a reducing agent, dithiothreitol (DTT), proteins are unfolded to their primary structure. With the above, proteins can separate based on molecular weight only.

Now back to your question...

2D-zymogram is a polyacrylamide gel which is co-polymerized with a protein substrate such as gelatin which will be used in our research application. It works based on the principles of SDS-PAGE which I have mentioned in my first paragraph. The addition of a protein substrate allows us to detect protease activity exhibited by proteases. Protease activity can be visualised as clear spots in the dark blue-stained gel.

As to your second question, let me define the two temperatures. 37°C is the normal human body temperature and 28°C is the environmental temperature. As I have mentioned in my post, S.maltophilia can be found in the environment. In order for S.maltophilia to infect an immunocompromised host, it must be able to adapt to the normal human body temperature and express its virulence factors. It was suspected that some of the periplasmic proteases found in the periplasmic space of the bacterium might be the virulence factors which account for its pathogenicity. Hence, if we observed that protease activity is higher at 37°C as shown by larger clearings in the gel as compared to 28°C, we can infer that the bacterium is able to express its virulence factors and increase its level of activity at 37°C and thereby infect the host.

I hope I have answered your questions... :)

Han Yang
TG01

MadTechs said...

hey han yang!

may i know what will happen if there is the formation of water droplets from condensation? how will it affect your results?

and you mentioned about disinfection, and i assume that when it is not done properly, it will result in growth of unwanted bacteria? how do you differentiate it from the ones you want to grow and those resulting from contamination?

much thanks
Liyanah Zaffre
0607718D
TG02

tg01 group 2 said...

Hi Liyanah,

Thankz for reading my post!

The water droplets formed will make streaking on the agar surface difficult as it will be slippery. The streaks that you performed on the 'watery' agar surface might not be straight and the liquid on the agar surface may just carry the bacterium all over the agar surface and isolation of colonies might not be possible. The water droplets may be a source of contamination too.

This brings back to the principle of aseptic techniques. Whenever you bring any materials into and out of the biosafety cabinet, it is a good practice to swab the materials with 70% ethanol to disinfect the surfaces of the materials. This helps to ensure sterility of the materials brought in and out of the BSC. This also applies to our agar plates too. When we have performed our streaks and intend to incubate, we should swab the agar plates with 70% ethanol (spray at a distance) before incubating.

In addition, the lab we used to culture Stenotrophomonas maltophilia belongs to biosafety level 2. In this lab, there is only S.maltophilia but not any other bacteria. When performing our streaks, we ensure that only 1 vials is brought into the BSC 2 (recall Mammalian Cell Technology)to prevent cross-contamination. So far, we have not seen any growth of unwanted bacteria given our strict aseptic techniques practiced as mentioned above. I guess spraying ethnanol at agar plates isn't that difficult right?

Thankz! :)

Han Yang
TG01

hellomedtech said...

Hey Han Yan,

When you identify the Stenotrophomonas maltophilia, how do you know that it is Stenotrophomonas maltophilia and not any other organisms? Like what characteristics as in colour or whether the colonies are concave or flat, etc..does the Stenotrophomonas maltophilia have?

Thank You

Dyana
0605169B

De Incredibles said...

hey

ooo ic. thanks for ur comprehensive explanation =P

Zhenling

tg01 group 2 said...

Hi Dyana,

:( My name is spelt wrongly...anyway...

The colonies of S.maltophilia are white/pale yellow in colour. They appeared round to me and they are smooth and glistening (do not ask me why they are smooth and glistening).

Hope that answer your question...!

Thankz!

Han Yang
TG01

tg01 group 2 said...

To Hanyang

hi, what does nosocomial means?

From ivan.

THE CODEC 5 said...

Hi Han Yang,
Sorry to hear bout the change of MP. It's a huge pity for the previous was really interesting. But this doesn't sound all too bad either eh.

Anyway, have got a question. Under "Why study S.maltophilia?" you'd mentioned these two points:

1.Ageing population
2.Increased use of surgical equipments

Could you care to elaborate on these please? Am not really sure how they relate to why 'we should study these microbes. Thanks!

Alexander Soo TG02
0608122H

group1 said...
This comment has been removed by the author.
group1 said...

hello han yang!

you mention that S.maltophilia isolates is one of your materials. how do you get them? where are they found? (as in from what specimens)

Thanks
Yu Mei
TG01

tg01 group 2 said...

Hi Ivan,

Thankz for reading my post!

Nosocomial refers to hospital. Nosocomial pathogen means hospital-acquired pathogen. Nosocomial infection refers to hospital-acquired infection. Hope you can understand!

Hi Alex,

Thankz for your interest in my post!

As we age, our immune system starts to deteriorate. This is the golden opportunity where the bacterium can invade into the host and infection can arise. Remember that I mentioned in my post that this bacterium infects immunocompromised individuals especially? As our population starts to age, we will become more susceptible to this bacterium infection. Hence, this urges a need to research on this bacterium so that appropriate drugs can be developed to target against S.maltophilia-associated infection.

S.maltophilia is found to colonize on surgical equipments. If strict aseptic techniques are not practiced to ensure the sterility of equipments, S.maltophilia can grow on these equipments. These equipments can serve as carriers by which the bacterium can invade into our body and cause infection. Since medical technology is advancing rapidly, there will be increased use of surgical equipments. This will give more opportunity for the bacterium to invade into humans if sterility of equipments are not taken care of. Hence, the frequency of S.maltophilia-associated infection will increase.

Hi Yumei,

Thankz for reading my post!

Majority of S.maltophilia isolates are obtained from ATCC (American Type Culture Collection). Some isolates are obtained from patient specimens from local hospitals. S.maltophilia can be commonly isolated from respiratory tracts and skin(wounds)

Hope I have cleared everyone's doubts! =]

Han Yang
TG01

BMT said...

hi han yang. what do you mean by ageing population? What does it have to do with s maltophilia?

thanks!

elyana
tg01
0606676e

BMT said...

Hi...can i know how you streak your agar plates to obtain isolated colonies?thanks:)

Rachael
Tg01

De Incredibles said...

Hi Han yang,

I believe there are many strains of S.maltophilia present. Can u share why these strains are chosen for ur project?

Also, what are the current solution to nosocomial infections?like how docs tries to get rid of this bacteria..is heat sterilization etc able to kill them.


Thanks

Jean Leong
TG02

tg01 group 2 said...

Hi Elyana,

Thankz for reading my post!

Ageing = growing old
Population = group of humans

Ageing population = population which is growing old

As we grow old, our immune systems become weaker, thus being more susceptible to bacterial or viral infection, with no exception to S.maltophilia infection. Hence, there is a need to study and understand more on this bacterium (e.g. identify virulence factors) so as to develop drug therapy for treating S.maltophilia-associated infection. Your question is somehow similar to Alex's. You may want to refer to my reply to Alex. Thankz!

Hi Rachael,

I perform a total of 5 streaks on the agar plate. Each set of streak contains 4 streaks (2 long and 2 short streaks) without touching each other (except for the first and last set of streaks). The 1st set of streaks is performed normally. The 2nd set of streaks is performed by streaking from the 1st set of streak. The 3rd set of streaks is performed by streaking from the 4 streaks of the 2nd set of streaks. The 4th set of streaks is performed by streaking from the 4 streaks of the 3rd set of streaks. The 5th streak is performed by streaking from the last 2 shorter streaks of the 4th set of streaks in a zigzag fashion to the middle of the LB agar plate. It is important to ensure the 5th streak does not touch the other 4 sets of streaks in order to obtain single isolated colony of S. maltophilia.

Pictures always explain better than words. You can always refer to the picture at this link...
http://www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/StreakingTechnique.htm

It is roughly similar to that picture except that the colonies are pale yellow in colour. :)

Han Yang
TG01

tg01 group 2 said...
This comment has been removed by the author.
tg01 group 2 said...

Hi Rachael,

The link I have provided is not complete, this is the final part of the link... http://www.life.umd.edu......... LabMaterialsMethods/StreakingTechnique.htm

Sorry for the inconvenience caused!

Han Yang
TG01

De Incredibles said...

Hi han yang,

u probably missed out my qns..

Hi Han yang,

I believe there are many strains of S.maltophilia present. Can u share why these strains are chosen for ur project?

Also, what are the current solution to nosocomial infections?like how docs tries to get rid of this bacteria..is heat sterilization etc able to kill them.


Thanks

Jean Leong
TG02

tg01 group 2 said...

Hi Jean,

Sorry for the late reply...

Yes, there are a total of 11 isolates/strains of S.maltophilia we are studying. These 11 strains are rather interesting to study as they are isolated from patient samples such as cerebrospinal fluid, blood culture, oropharyngeal region and more...In addition, these S.maltophilia are suspected to contain virulence factors which allow them to cause an infection. Hence, we would like to know more about the virulence factors which may aid in the development of vaccine (drug-specific targets) in the future.

Combinations of antimicrobial agents have been proposed to be effective against S.maltophilia although further studies are required. From the susceptibility tests conducted, it was found that S.maltophilia is susceptible to drugs of the tetracycline class. Vancomycin (antibiotic) remain to be the drug of choice when it comes to treating S.maltophilia infection.

As the saying goes, 'Prevention is always better than cure'. As S.maltophilia is known to grow on catheters and other surgical equipments, it will be advisible to sterilise and disinfect all surgical equipments before use. Good hand hygiene practices are strongly encouraged. Lastly, building up your immune system will keep S.maltophilia away from you as it seldom infects healthy individuals.

Hope I have answered your questions...!

Han Yang
TG01

Ms_chew said...

I enjoyed all the interesting questions posed to Han Yang. I think Han Yang's reply is also very clear and precise in answering the queries of his peers. Keep up the good job.