• 

When we entered the Biosafety and Environment laboratory, we went straight to the incubator and observed the culture media we inoculated yesterday. We observed what we expected that was that the last line of streaking had the thinnest lines and many isolated colonies of bacteria can be observed. These colonies can be further described by using some specific terminologies. First is the form or the basic shape of the colony such as circular, filamentous, etc. The size or the diameter of the colonies can also be measured using a ruler. Some also measure the side view of the colonies or their elevation viewed when the petri dish is turned on one end. On a closer look, the margin or the border of the colony can be magnified and observed as well as for its surface (smooth, glistening, rough, wrinkled or dull). Other colony characteristics that can be observed are its opacity (transparent or clear, opaque, translucent, etc. ) and the pigmentation or color (white, red, purple, etc.).The colonies we observed were similar in overall morphology which means that it is possible that they are of the same group. When colonies are distinct, each colony will represent an individual bacterial cell group.

• 

• 

We then proceeded to the microbiology room and made stock cultures of the colonies in our culture media. These will be kept so that the microorganisms will be in viable condition or so that there will still be a source of the microorganisms if they will be needed. We observed aseptic techniques such as flaming the loops and then dubbing it on to a bacterial colony. The inoculationg loop or needle is then stabbed on to a small tube containing a culture media and then they will be stored in the refrigerator. Also, some colonies were placed using inoculating needles in small microcentrifuge tubes containing double distilled water. They were then placed in a 95 degrees Celsius water bath and this is the Boiling Method of bacterial DNA extraction.

• 

• 

While waiting for the microcentrifuge tubes with bacterial colonies, we helped in testing some blood samples sent to the laboratory for Brucella spp. using the RBPT.

• 

• 

For each of the organ sample’s (lungs, liver, mesenteric lymph node) culture media, 2 representative colonies were taken so there were 6 total samples. After boiling them to extract their DNA, they were then used for PCR mixture preparation. The master mix consists of 56.8 ul of double distilled water, 20 ul of buffer, 40 ul MgCl2, 24 ul dNTP, 8 ul each of forward and reverse primers and 3.2 ul of Taq polymerase. Five microliters of DNA for each sample was then mixed with a corresponding amount of the mixture.

• 

The PCR tubes containing the PCR mixture were then placed inside the thermal cycler or the PCR machine and the run time was approximately 2 hours. Thermal cyclers are “DNA amplifiers” which regulate the temperature in different cycles (denaturation, annealing and extension). The following steps will not be done yet. Mam Noemi said that the PCR products will still be further processed, will be sequenced through an automated DNA sequencer and then the sequence results will be analysed by the BLAST search engine.

• 

Immediately after we entered the laboratory, we checked the culture media we incubated yesterday and observe who had the best streaking.

• 

• 

Mam Noemi then instructed us to make another culture media made from Potato Dextrose Agar (PDA) which is mainly for the culture of fungi. According to its label, it is actually a general purpose medium for molds and yeasts.  It is a recommended medium for plate counts in testing food and dairy products and in growing clinically significant organisms. It has a nutritionally rich base (potato infusion) which encourages growth, pigment production in some dermatophytes and sporulation in molds.

PDA is composed of dehydrated potato infusion and dextrose which encourages the growth of fungi which are usually hard to grow. Agar is the solidifying agent of the medium. We prepared the mixture by adding 500 milliliters of double distilled water to 19.5 grams of commercial PDA powder which contains 20 grams of dextrose, 15 grams of agar and 4 grams of potato starch.

• 

• 

• 

• 

During our lunch break, we visited one of the 12 regional centers of PCC which are strategically located nationwide, PCC- CLSU. Just like the PCC National Headquarters mission and vision, the center aims to help  improve the state of rural farming through carabao genetic improvement, technology development and dissemination, and establishment of carabao-based enterprises which will help ensure higher income and better nutrition.

• 

• 

• 

• 

• 

• 

Near PCC-CLSU was another center, the Small Ruminant Center which is headed by a different center director from that of PCC. They also aim to help develop, disseminate and commercialize technologies for the socio-political well-being, technical and economic needs, environmental concerns and cultural demands of the farmers. They continuously develop technologies, produce quality stocks and promote the goat and sheep industry in the country and Luzon in particular.

• 

• 

• 

When we went back to the laboratory, Mam Noemi instructed us to pour the agar mix we prepared to disposable plastic petri dishes. After we poured it, Mam Noemi closed the biosafety cabinet and turned on the switch for UV light. Mam Noemi said that that was to sterilize microbiological surfaces inside the cabinet as well as the materials and media inside the cabinet. This is an example of an Ultraviolet germicidal irradiation (UVGI) which is a disinfection method which uses short-wavelength ultraviolet(UV-C) light for killing or inactivating microorganisms. UV light can destroy the nucleic acids and thereby disrupting bacterial DNA, leaving them unable to perform vital cellular functions and killing them.

• 

• 

While waiting for the decontamination, we helped the NVSU interns test the blood samples from the Gene Pool for trypanosomes using a simple microtechnique. The technique uses centrifugation of blood in a capillary tube and observing for trypanosomes in the buffy coat. Other blood protozoa may also be observed through this technique. One of its advantages is that the technique does not alter the morphology of the trypanosomes. The only difficulty we encountered with this technique is that the diffraction of light casts shadows at both sides of the capillary tube and therefore it is difficult to examine the sides. This may reduce the capacity of the technique to detect low numbers of trypanosomes especially if they are at or near the sides.

• 

We also helped sort out and check all the fecal samples indicated on a list by Doc Chat.

• 

• 

• 

• 

• 

Mam Noemi acquired the plates she decontaminated in the biosafety cabinet through UV irradiation then we proceeded to the corridor. Mam Noemi said that the occupational health nurse requested in giving them additional health awareness by conducting swab testing to determine the amount of microorganisms present in some equipment (e.g. finger scanner, office door knobs, stair handles and telephones) which many of the employees are using. This simple effort is important since the results will help them create a more suitable preventive action to prevent the spread of germs.

Mam Noemi showed us the materials we need for the activity, some sterile cotton swabs, 9 ml of phosphate buffered solution in sterile conical tubes. We first did Equipment Swabbing by rubbing the swabs back and forth with heavy pressure on some of the equipments in the offices and in the hallway common to all (e.g. office door knobs, finger scanners, telephones, printers, doorbell). We then immediately returned the swabs in dilution fluids and labeling them thereafter.

Next thing we did was to test for the Air Quality to assess if the air within the environment is free from contaminants. We made use of the PDA plates for this test by opening the cover of the culture media and placing the petri dishes at random locations in each room for 15 minutes.

• 

For our final activity for this day, we helped the NVSU interns process some blood samples for PCR of Surra.

• 

• 

• 

• 

As we entered the Biosafety and Environment Laboratory, sir Dadz tasked us with the fecalysis of fecal samples from a goat herd. We did the usual routine sedimentation and flotation techniques of fecalysis in the laboratory together with Jakee and Jendy. We observed many Toxocara spp. and Strongyloides spp. ova in one of the fecal samples.

• 

• 

After our fecalysis, we went on to observe Mam Noemi who was preparing different types of culture media. Culture media are important in microbiological tests since they help grown and obtain pure cultures, count microbial colonies, and cultivate microorganisms selectively. It increases the possibility of achieving accurate and repeatable test results.

These culture media contains gelling agents (agar), nutrients, energy sources, growth promoting factors and other elements such as minerals and salts depending on the type of culture media. There are several categories of culture media sucg as growth media which are designed to grow most bacteria. Other categories include transport media for preserving bacteria during transport and enrichment media which increases the numbers of desired bacteria.

• 

• 

• 

• 

We helped Mam Noemi in making cultures of microorganisms that we were trying to isolate from organs of deceased water buffaloes submitted to the laboratory for disease diagnosis. The three culture media we used today for that purpose were Mannitol Salt Agar (MSA), MacConkey Agar and Eosin-Methylene Blue (EMB) Agar.

We helped Mam Noemi in making cultures of microorganisms that we were trying to isolate from organs of deceased water buffaloes submitted to the laboratory for disease diagnosis. The three culture media we used today for that purpose were Mannitol Salt Agar (MSA), MacConkey Agar and Eosin-Methylene Blue (EMB) Agar.

                MSA is a selective medium which contains a high concentration of salt (7.5 %) that is inhibitory to most bacteria. It is also a differential medium in that it contains the sugar mannitol and phenol red. Bacteria which are able to ferment mannitol will produce acid turning the red media to yellow (indicative of pathogenic species). MacConkey Agar and EMB are also selective and differential media only differing in components. MacConkey Agar contains bile salts and crystal violet which inhibit the growth of most gram positive organisms. It distinguishes non-lactose fermenters from fermenters due to the presence of lactose and neutral red as the pH indicator. The media also contains gelatin and peptones that prove the essential nutrients for growth. EMB, on the other hand, contains eosin and methylene blue which inhibits gram-positive bacteria and lactose and methylene blue as pH indicator.

Mam Noemi taught us proper streaking techniques. Proper streaking is necessary in isolating a pure strain or species of bacteria. It is ideally a rapid and simple process of dilution isolation. It can be done by using any sterile tool such as a cotton swab but in the laboratory they use inoculation loops and needles. To prevent contamination of the agar plates, we did the streaking inside a biosafety cabinet.

The inoculation loop was first sterilized by placing it inside an incinerator. When the loop is cool, it is dipped in a media containing a piece of the organ sample where many bacterial colonies are present. The inoculation loop was then dragged across the surface of an MSA, MacConkey Agar or EMB Agar back and forth in a zigzag motion until ~30% of the plate was covered. Streaking was done in four directions wherein the heaviest growth should be observed in the first section and the last would have many isolated colonies. The plates were then placed inside the incubator and will be observed the following day.

• 

• 

For today, we were assigned to Doc Jega at the Reproductive Biotechnology Laboratory and Physiology Unit of PCC. They conduct reproductive technologies there which are helpful tools for the improvement of the herd and also in multiplying desirable characteristics of the herd’s progeny. However, since Doc Jega has still some matters to attend to, we helped NVSU interns Jakee and Jendy first prepare some blood samples submitted at the Biosafety and Environment Laboratory for DNA extraction prior to PCR.

• 

• 

When Doc Jega arrived, we headed down to the second floor where their laboratory was located. Doc Jega introduced us to BioPRYN^®, a test which offers an alternative and easier way for the confirmation of pregnancy in beef cattle. PCC is testing if this accurate test for pregnancy may be applied to water buffaloes and if they can use it for an early re-breeding program. The test detects for the presence of Pregnancy-Specific Protein B (PSPB) which is a protein in the blood circulation of an animal that is only produced by the placenta containing a growing fetus. In addition, Doc Jega said that it is effective in pregnancy detection as early as 28 days as compared to ultrasonographic detection of pregnancy which is 30 days.

Doc Jega acquired some samples for the test so we can try it. Before starting with the assay, the kit reagents as well as the BioPRYN Assay Plate (PL) were prepared at room temperature. For the assay proper, the first step done was by adding a detector solution using a multi-channel pipette to each well of the PL. Standards were added next using a fresh tip for each solution and were run in duplicate (a Standard Hi and a Standard Lo). The third step was bu adding a sample serum to each of the remaining wells using a fresh tip for each sample. The position of each sample was verified using a printed Plate Grid which goes with the kit. After all those steps were done, the microplate lid was sealed with ParaFilm and was incubated for 2 hours at room temperature (18-24 degrees Celsius). While waiting for those 2 hours, we went out to have lunch.

• 

• 

• 

• 

After we had our lunch, we decided to visit a nearby center which is the National Freshwater Fisheries Technology Center (NFFTC) of the Bureau of Fisheries and Aquatic Resources (BFAR) located within the Central Luzon State University (CLSU) campus. According to the center’s history, it was established in 1979 as a joint project of BFAR, United States Agency for International Development (USAID) and the Texas A & M University (TAMU). It functions according to the Philippine Fisheries Code of 1998 (RA 8550) to serve as a Philippine Germplasm Reference Collection Center for Tilapia and other freshwater species. It establishes and maintains a collection of a variety of freshwater species in the Philippines as well as from other countries for development of broodstock for future use. Those working at the center also helps in the development and evaluation of different studies on aquaculture production system techniques. 

• 

• 

When we came back to Doc Jega in the afternoon, we continued the BioPRYN test. The ParaFilm seal was removed and the contents of the wells were removed by striking the inverted plate on a tissue paper (blot drying). The microplates were then loaded in an automatic plate washer machine which washed the plate four times with distilled water. Blot drying was again done after. Then, a second solution was added to the plates which is an enhancer solution. It was added using a multi-channel pipette and then the plate was incubated for 30 minutes after covering it with ParaFilm. Blot drying and washing four times were again done and then a TMB substrate solution was added to each well.  The plate was then incubated for 15 minutes at room temperature. After incubation, a stop solution was added to each well using a multi-channel pipette and the well contents were gently mixed through gently swirling for few a times. The plates were read between 630 and 650 nm using an ELISA plate reader.

• 

• 

• 

• 

Doc Jega decided to show to us other laboratories of PCC and so we went on laboratory hopping. Our first stop was at the Nutrition Laboratory which is laboratory supporting the research and development activities of PCC. They also provide technical services and information to farmers by the assessment of the nutritive value of feedstuffs and other related samples. They also collaborate with other researchers and even students with regards to new knowledge and information. They help address nutritional problems and deficiencies of ruminants (especially the water buffaloes at the Gene Pool) and other livestock species.

One of the laboratory personnel who were touring us said they perform a test that is familiar to us which is proximate analysis. Components of feed such as dry matter/moisture, ash, crude protein, crude fat and crude fiber were being measured. They do detergent analysis such as acid detergent fiber, neutral detergent fiber and acid detergent lignin and mineral analysis such as Calcium and Phosphorus and other relevant analysis.We met sir Reddik who was getting the measurements of some feedstuff acquired from fistulated water buffaloes at the Gene Pool.

• 

• 

• 

• 

Beside the Nutrition Laboratory was the Dairy Laboratory which provides services for the analysis of milk components.  It supports the Genetic Improvement Program of PCC. Their services are mainly intended for dairy herds who are participants in the milk recording and performance testing of PCC such as their assisted cooperatives. In contrast to the California Mastitis test which is a subjective test, milk samples are rapidly being analyzed by a milk analyzer for quantitative components such as milk fat percentage, milk protein percentage, lactose, total solids, and somatic cell count (SCC).

According to Mam Paulen, one of the staff working in the laboratory, analysis is usually completed within nine working days from the date of request or submission of milk samples. They can, however, preserve the raw milk samples for up to 7 days at 4-5 degrees Celsius by adding bronopol.Unpreserved samples were immediately placed in a container with ice and analyzed within 2 days or 72 hours after its collection. Farmers were also oriented or trained regarding the proper sampling of milk, handling and storage.