Microbiology

Topic: Mycology
Procedure: Fungal Culture
Posted by: Azhar Hamdan (0503269C)

In this post, I am focusing on the fungal culture procedure for clinical specimens.

Purpose

Fungal cultures on clinical specimens are done to detect the presence of:
• Yeast
• Yeast-like fungi
• Hyaline or dematiaceous filamentous fungi
• Aerobic actinomycetes

Common specimens received
• Cerebrospinal Fluid (CSF)
• Blood
• Vaginal/Cervical Swabs
• Skin
• Hair
• Nail

Media Used
• Sabouraud Dextrose Agar (SDA)
• Sabouraud Dextrose Agar with Chloramphenicol (SDC)
• Brain Heart Infusion Agar with Chloramphenicol and Gentamicin (BHICGA)
• Brain Heart Infusion Agar (BHIA)
• Mycobiotic agar with Actidione (AA)
Chloramphenicol, Gentamicin & Actidione are antibiotics. The purpose of adding antibiotics to media is to suppress normal flora and thus to allow for the multiplication and detection of the pathogenic organisms



Okay, maybe all the media looks the same, but just to point something out, different media have different corresponding coloured covers. This prevents accidental mix up.
(Left to right; SDA, SDC, BHICGA, BHIA & AA)



General Procedures
• The ideal temperature for the recovery of fungi is 30°C
• Cultures are kept for 2-4 weeks depending on the specimen and the suspected organism before cultures are reported as negative and then discarded
• Media is preferably prepared as a slant in a tube as opposed to being prepared in a Petri-dish

Reasons for preference of slant:
• Potentially less hazardous for staff to work on ( e.g. due to the vertical tubes, aerosols produced would less likely escape)
• Less prone to contamination and dehydration
• Easier to store in racks



Pictures were taken with permisson from the supervisor.

Thats all for this post. Say HELLO to week 10

Lab Techniques

Hi everyone,

In this post, I am going to talk about 2 imagers, PharosFX™ Plus and VersaDoc MP 4000. Both the imagers are from Bio-Rad. I will focus more on PharosFX™ Plus as I will be using it for my MP. After running a 2D gel (refer to Jiaxin’s post) and before identifying the proteins (refer to my previous post and Shahirah’s post), the gel image will be acquired using an imager and analysed with a software.


PharosFX Plus main unit, external laser and computer with software


Sample tray to place the gel

The pictures are taken from http://www.bio-rad.com/B2B/BioRad/product/br_category.jsp?BV_SessionID=@@@@1782679805.
1187184231@@@@&BV_EngineID=ccchaddlkejjjhgcfngcfkmdhkkdflm.0&divName=Life+Science+Res
earch&loggedIn=false&serviceLevel=Lit+Request&lang=English&csel=SG&catLevel=6&catOID=
-35087&isPA=false&categoryPath=%2fCatalogs%2fLife+Science+Research%2fImaging+Instrume
nts%2fImaging+Instruments+by+Application%2fColorimetric+Detection%2fMolecular+Imager+
PharosFX+Plus+System

PharosFX™ Plus consists of a scanner, a 532nm internal laser and 7 emission filters. As our experiment would also require wavelengths of 488nm and 632nm, an additional external laser was required. The different lasers will excite the respective fluorescent dyes to fluoresce. The wavelength of the fluorescence emitted will be in a range so emission filters are required to filter out other wavelengths, obtaining the desired wavelength. The scanner will then scan the gel from top to bottom and image is obtained. For different applications, different trays are used. For gels that are less than 3mm thick, the default tray (Sample Tray) is used. For microplates and gels that are thicker than 3mm, Multisample Tray 1 is used. For gels that are sandwiched between glass plates, Multisample Tray 2 is used.

Unlike PharosFX™ Plus, VersaDoc uses camera to acquire gel images. Both imagers have their pros and cons. The use of either imager would depend on the application or the type of stain used.

Application

One application of PharosFX™ Plus is that samples (eg. Protein, DNA) in microplates can be quantitated. However, the Sample Tray must be changed to Multisample Tray 1 in order to place the microplate. One application of VersaDoc is that it can be used to do colony counting. Even so, we had to count the colonies manually because we are not able to bring the bacteria out of the BSL.

Type of stain

For fluorescent-, colorimetric- and isotopic-stained gels, it is recommended to use PharosFX™ Plus because in VersaDoc, there is a certain distance between the camera and the gel. The space in between will affect the intensity of the signal captured. With the use of PharosFX™ Plus, the intensity of the signal is fully captured as the gel is in direct contact with the scanner.

For chemiluminescent and ethidium bromide-stained gels, it is recommended to use VersaDoc because PharosFX™ Plus uses scanning (from top to bottom) to acquire the gel image. By the time the scan reaches the end, the signal at the bottom of the gel would have decreased (rate would depend on the half-life of the compound). If VersaDoc is used, the signal will be captured as a whole without any time difference between different points on the gel.

After acquiring the gel image, a 2D analysis software, PDQuest is used to analyse the proteins quantitatively (up/down-regulation) and qualitatively (absence/presence). Quantitatively, we would detect the difference in the intensity between protein spots that are in the same position on two different gels. Usually, only a more than 2-fold increase or decrease will be considered significant because if the difference is only a little, most probably it might be due to system variation (eg. difference due to variation between different machines used) rather than biological variation (difference due to physiological change or drug treatment). Qualitatively, we would detect protein spots that are present in one gel but absent in another gel.

That's all for this post!

Ming Boon
0503197F
Tg01

Peripheral and Fetal Cord Blood Cultures

In prenatal diagnosis, blood karyotypes are usually done for birth defects, mental retardation, infertility or multiple miscarriages. Stimulated peripheral and fetal cord blood cultures are used to establish the constitutional karyotype of an individual. The procedure is based on the concept that every cell in the body contains the same complement of DNA as any other cell. This is true for most cases; an exception occurs in mosaicism, in which more than one cell line is present in an individual. It is convenient to study chromosomes of peripheral blood lymphocytes as it is easily obtained via venipuncture while fetal cord blood is obtained from the umbilical cord. Blood specimen usually produces long chromosomes and consistently obtains chromosome preparations of good quality.

Lymphocytes in the peripheral blood rarely undergo spontaneous cell division, thus it must be ‘stimulated’ to grow and divide in vitro. Various mitogenic agents are available to stimulate B or T lymphocytes; the most common agent is phytohemagglutinin (PHA), which has a stimulatory effect primarily on T lymphocytes. T lymphocytes account for approximately 85% of the circulating blood lymphocytes and are nearly always present in peripheral blood.

With respect to newborn (< 3 months) and fetuses, a preliminary result is usually available within 3 working days by utilizing a 48hr blood culture, adapted from the 72hr blood culture and harvest procedure. Such method is best utilized for cases of possible aneuploidy ( extra multiple of the haploid number of the chromosomes, eg. Down syndrome, extra 21). The 48hr preliminary result is then followed up with the 72hr result that provides longer chromosomes with higher banding resolution. The difference between 48hr culture and 72 hr culture is, 48hr culture adds EB instead of thymidine, usually being to 72hr cultures.

Culture set-up

Specimens are set up as suspension cultures. To increase the number of metaphase chromosomes, thymidine or methotrexate (MTX) may be added to synchronize the cultures.

After an optimal culture time, the addition of a mitotic inhibitor such as Colcemid® causes the chromosomes to contract and condense, ethidium bromide (EB), a DNA-intercalating agent, may be added to the cultures. When added at G2 in the cell cycle, this interferes with normal condensation of the chromosomes.

The addition of EB during harvesting period results in extended chromosomes and an increase in band resolution. Slide preparations of metaphase chromosomes are obtained following exposure of the culture to a hypotonic solution (Ohnuki’s) to spread the chromosomes apart, followed by a series of fixative (3:1 methanol : acetid acid) steps.

To make slides, 3-6 drops of the cell suspension from a glass pipet along the upper edge of the slide are dropped from height of 10-20cm, depending on the spread, while holding the slide horizontally or vertically to the bench at a 45° angle. Firmly bang the slide on the bench top several times before placing it on a 56°C warming tray. This may help in chromosome spreading. However, do not bang if there is widespread scattering.

Check the spread under the phase contrast microscope before continuing with rest of the slides. Do the appropriate adjustment of the cell density or height to achieve the best spread subsequently. Bake slide at 90°C over for 2 hours, or at 60°C overnight and a further 1hour at 90°C the following morning.


Lau Yi Fang Yvonne
0503149G
TG01

Lab Techniques

Hi everybody! I'm attached to a research center which all of you are VERY familar with. =D My MP and SIP are done together with Ming Boon, Johanna & Shahirah. Our research revolves around proteomic (protein-related studies) work and the bacteria that we are looking at is Stenotrophomonas maltophilia. It is a Gram-negative bacillus which causes nosocomial infections. We are working on 2 different strains, the environmental (from soil) and clinical (from patient infected with S.maltophilia). Hopefully, we will be able to identify the difference in protein composition between the two strains, which makes the clinical strain infectious.

As you know, a cell is made up of many different kind of proteins; secretory, cell membrane, cytoplasmic and etc. So, it would probably take years to study and analyze all of them! We were given a visable goal which is to look into only 2 types of protein; secretory and exposed cell membrane proteins. These 2 types of proteins are selected as a microbe usually attaches to and infects a cell through secreting proteins or sticking onto it through the exposed cell proteins. At this point of time, we are still analyzing the secretory proteins.

The secretory proteins alone consist of many different proteins. In order to analyze individual proteins, they first have to be separated. For our research we're using the 2-Dimensional Electrophoresis approach.


First Dimension Separation : Isoelectric Focusing (IEF)

Proteins are amphoteric molecules such that they can carry either positive, negative or zero net charge depending on the pH of their local environment. For every protein, there is a specific pH at which its net charge turns zero. This is its pI. pI refers to isoelectric point and is defined as the pH at which a protein will not migrate in an electric field and is determined by the charges it carries.

The proteins are placed in a medium with a pH gradient. When electric field is applied, they will move towards the electrode with opposite charge (unlike pole attracts). During migration through the pH gradient, proteins will either gain or lose protons leading to decrease in net charge and mobility. Ultimately, the protein will reach a point where the pH gradient is equal to it's pI. Being uncharged, migration would stop. In any case where a protein were to diffuse to a pH region lower than its pI, it will be protonated and forced back towards the cathode by the electric field. On the contrary, if it diffuses to a region of pH gradient greater than its pI, the protein will be negatively charged and move towards the anode. Through this way, focused spots are obtained.

P.S. The circles represents the proteins and the number
within it is its pI

Adapted from: http://www.bmskorea.co.kr/bms_product/bms_Product_Sec/bms_Product_Sec_List.aspx?sec=sec&cstep=3&cgroup=32

For our project, the pH gradient used is a strip of acrylamide matrix gel that has pH gradient incorporated covalantly making it immobolized even under electric field. These strips will have to be rehydrated with rehydration buffer and protein samples, together with the buffer, the proteins to be separated will be absorb into the gel strip. The gel strip used is called IPG strip.


Second Dimension Separation : SDS PAGE (Molecular Weight)

The pH gradient strip is then placed in the well of the acrylamide gel for 2nd dimension separation.

The proteins on the pH gradient gel migrates down, separation based on molecular weight.

Reminder: The numbers refers to protein's pI

The black spots indicates the stained and separated proteins

Adapted from: http://www.biosciencetechnology.com/ShowPR.aspx?PUBCODE=090&ACCT=9000012495&ISSUE=0401&RELTYPE=PR&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;PRODCODE=00002750&PRODLETT=B


The protein spots will be excised with Xcise (scroll down to view Ming Boon's post) and analyzed with MALDI (scroll down to view Shahirah's post).

THAT'S ALL for my post! =D


Tang Jiaxin
0503257H
TG01