Week 12

Hi, this week I'll be sharing on microbiology.

In the microbiology lab, the most commonly received types of specimens would be urine, stool and swabs from any part of the body for culture. Culture basically refers to specimens being plated on specific agar/s to observe for any predominant growth of microorganisms. If culture results confirmed that the patient is infected, CDS (calibrated dichotomous sensitivity) test would be done to determine the antibiotic sensitivity of the microorganism in order for doctors to prescribe the correct type of antibiotic to the patient.

CDS (another term for antibiotic sensitivity) Test

Stool specimens are one of the most frequently received specimens in the lab. Stool cultures would be able to show the presence of certain types of abnormal microorganisms such as Samonella, Shigella, Campylobacter and Vibrio species present in the normal human body. For oncology patients, stool screening is required to detect any multi antibiotic resistant microorganisms such as ESBL producers, VRE or any unusual overgrowth of pathogenic flora such as P. aeruginosa or Candida.

Because of the fact that human stool is not sterile, there is a high possibility that there would be a mixture of gram positive or negative, aerobic or anaerobic bacteria present. Therefore, differential media (eg. XLD, TCBS and Campylobacter agar) need to be used to differentiate the normal from pathogenic flora present in the stool.


Besides stool cultures, high vagina swabs (HVS) are also very commonly received in the lab. Because I work in a gynae hospital, most of the HVS received are to detect for Group B Streptococcus in pregnant women. Although group B Streptococcus can be considered as a type of normal flora in the female genital tract, the presence of this organism in pregnant women is clinically significant.

Swabs used for Culture
Retrieved from http://home.caregroup.org/departments/pathology/lab_manual/PLM_containers.htm

Group B Streptococcus can cause a relatively rare but serious infection in newborns. Approximately 10-30% of pregnant women carry this type of bacteria in the vagina or rectal area, and may pass in to their babies during labour. Some of the consequences include sepsis, pneumonia or meningitis in newborns. Some of these babies, particularly those with meningitis, will have long-term health problems such as hearing or vision loss, cerebral palsy, or developmental disabilities and about 5% would not be able to survive. Therefore, to reduce the risk of the infection in newborns, prenatal testing and treatment is often recommended.

Processing:
1. Swab would be plated on blood agar and put into TGN enrichment broth.
2. Plates and broths are then incubated for 37°C for 24 hours.
3. After incubation, sub-culturing is done by plating the TGN enrichment broth on blood plates.
4. Original blood plates incubated for 24 hours will be checked for organisms such as strep B, Candida and Listeria.
5. Subcultures will be read the following day for strep B.

For all types of suspected microorganism growths on cultures, biochemical tests must be carried out to confirm their identity. Different types of mediums are used to identify the characteristics of the microorganisms based on their biochemical reactions. Below are some examples of mediums that are commonly used (brief explanation):

Kliger Iron Agar (KIA) – Detects the ability of the microorganism to ferment glucose by observing the reaction produced be the organism in the agar. KIA also contains sodium thiosulfate to test for hydrogen sulfide. Iron salts present in the media react with the hydrogen thiosulfide to produce an insoluble black precipitate.

Citrate Test (Simmon Citrate Agar) – Determines the ability of the microorganism to use sodium citrate as the sole source of carbon for metabolism and growth.

Urease Test - Urease is an enzyme that breaks the carbon-nitrogen bond of amides to form carbon dioxide, ammonia, and water.

OF (Oxidation Fermentation) Basal Media – Used to determine oxidative and fermentative metabolism of carbohydrates by gram-negative bacteria on the basis of acid reaction in either the open or closed system. Closed system refers to the surface of the agar being covered with oil.

Motility Indole Lysine Medium – Used to demonstrate motility, indole production, lysine decarboxylase and deaminase activity and hydrogen sulfide production in microorganisms.



Ka Hang
TG02

Double posting! (1st part: Simple Introduction to Flow Cytometry)

Hi all

I had gotten some feedbacks from some of our fellow course-mate that my entries are a little too technical and very hard to understand. So I decided to skip the technical stuffs and go straight in to the topic with simple explanation.

So feel free to ask me any questions regarding any parts that you do not understand in my entry.

Simple 4 steps explanation of how an experiment with the Fluorescent Activated Cell Sorter (FACSAria), also known as a flow cytometers, works:

1) Load the samples onto the FACSAria
2) Data will be acquired
3) Set gating to select out the cells of interest
4) Record the data

Flow cytometry is simply just to analyze the data acquired from cell population that was loaded onto the FACSAria. It analyze by detecting and identifying the cell population of interest that were set aside differently from rest of the cell populations.

Some examples of how this could be done are through the uses of cell markers and certain dyes, like fluorescent-coupled antibodies or fluorescent dyes or fluorescent proteins.

Example of Fluorescent coupled antibodies:
1) Antibodies against CD44 cell marker, coupled with PE fluorochrome

Note: PE stands for phycoerythrin

Example of Fluorescent dyes:
1) Hoechst 33342

Example of Fluorescent proteins:
1) Green Fluorescent Protein (GFP) via cell transfection

Okay, that’s all to my introduction to the flow cytometers and I will stop any further complex explanation here. Hope everyone could have a rough understanding of what flow cytometry is about.


Many thanks
Quan Jun
TG02
Group 08
08 September 2008

Double posting! (2nd part: SP Profiling in Stem Cells)

Up next is my introduction to my Major Project (MP), which is related to Side Population (SP).

Short Introduction

Side population is a defined population of cells that could be distinctly identified from the rest of the heterogeneous cell population with the use of Hoechst 33342 dye in the flow cytometers. Using defined filters that allow the collection of the emission profile of the used dye does this.


The primary objective of my project is to identify the best blockers blocker and its optimal concentration in blocking ATP-binding cassette (ABC) family of transporters. The blockers are Verapamil, Fumitremorgin C and Reserpine respectively.

We will be using bone marrow cells.

ABC family of transporters is responsible for the high efflux of the Hoechst dye that contributes to the low Hoechst staining which could be identified with the use of flow cytometers. We use isolated bone marrow from murine species for SP identification.

Below is a diagram that shows how SP cells are identified and gated.








The following is the protocol in which my laboratory follows to acquire SP profiling in flow cytometry.

(The protocol is only for understanding purposes; so certain information is left out, please understand)


DMEM = Dulbecco’s Modified Eagle’s Medium

HBSS = Hank’s buffer salt solution (Hank’s Balanced Salt Solution)

Protocol (Flow chart):

Euthanize the mice/rodent

Use surgical instruments to remove the fur from the fore and hind limbsRemove and store the fore and hind limbs in cold DMEM+(with EDTA)
Transport the specimens on ice and process in the laminar flow hood
Remove the meat from the fore and hind limbsCut the ends of the femur, tibia, humeral and radius

Flush the interior of the bone until it turns white
Filter the cell suspension obtained with a 100um cell strainer
Centrifuge the cell suspension
Remove all but 500ul of the supernatant
Add erythrocyte-lysing solution in a ratio of 11:1

Incubate for 5 minutes under room temperature
Centrifuge the cell suspension
Remove the supernatant and wash 2 times with cold HBSS+
After washing, centrifuge the cell suspension again
Remove the supernatant and add 2ml of cold DMEM+(without EDTA)

Count the number of cells using a hemocytometer
Calculate the total number of cells in the cell suspension

Prepare and label empty 15ml falcon tubes

(The tubes were labeled “unstained”, “PI only” and “Hoechst + PI”, “Hoechst + blocker + PI”)

Pipette in 1mL of pre-warmed DMEM+(without EDTA) in each of the tubes
Calculate the amount of cell suspension to add to achieve a final concentration of 106 cells/ml for each of the tubes
Place the tubes in the waterbath (37oC)
Add blockers to “Hoechst + blocker + PI” tubes according to the blockers to be used

Pre-incubate the cell suspension for 15mins at 37oC
After pre-incubation, add in the Hoechst 33342 dye to all the tubes and incubate for a further 90mins at 37oC

(Except for “unstained” and “PI only” tubes)
Mix the cell suspension at every 20mins interval during the incubation
After incubation, centrifuge the cell suspension
Remove the supernatant and resuspend in cold HBSS+ with PIStore on ice and out of light before data acquisition on FACSAriaRecord the SP profile acquired

That's it for my entry this week.

So as to not confuse anyone, the protocol was briefly explained, so feel free to ask me questions if there is any doubts.

Many thanks

Quan Jun

TG02

Group 08

08 September 2008