Lab 2 Discussion There are 2 methods of obtaining pure cultures from samples containing mixed populations.

One such method is the dilution and spread plate technique. This method is often used when the mixed populations of prokaryotes exist in a natural sample (i.e. soil, pond water, fecal material,etc.) and the researcher would like to obtain isolated colonies and enumerate the number of culturable prokaryotic cells in the sample. The other method of obtaining pure cultures is called streak plate. This method is mostly used to separate mixed populations of prokaryotes in culture (growing culture media).

In this experiment, we used serial dilution and spread plate technique to obtain the isolated colonies of bacteria that exists in porridge. Because of their very small size, counting the number of bacteria in porridge can be difficult. Therefore, we used serial dilution and spread plate technique to determine the number of bacteria colony in the porridge. We make a wide range of dilutions of original sample (porridge) to get countable numbers of bacteria in the sample. The porridge is diluted into the diluted factor of 10-2, 10-3, 10-4 and 10-5. Therefore, the high number of bacterial cell can be reduced and easy to count. From our result, the broth culture that contain the porridge sample have changes. The broth culture become less cloudy from the 10-2 diluted factor to 10-5 diluted factor. It also means that the concentration of the bacteria in broth culture is reduced. This shows that the number of bacteria has reduced by using the serial dilution. Moreover, the growth features of bacteria in all broth culture are show pellicle on the surface of broth culture and in flocculation condition.

For spread plate, the number of bacteria colonies is decreased from the 10 -2 diluted factor to 10-5 diluted factor. The number of bacteria colonies in the dilution 10-2,10-3 and 10-4 spread plate are more than 300 colonies. This is due to the bacterial concentration is too high. While the dilution 10-5 spread plate has 256 colonies. By using the formula, the total number of bacteria colonies in the porridge is 2.56108 CFU/g.

By using gram staining, the type of bacteria that exists in the porridge can be identified. We used the bacterial colony in the spread plate for staining. From the gram staining, there are two type of bacteria that exist in the porridge. There are Bacillus cereus and Escherichia coli. Under microscope, Bacillus cereus is rod-shaped and apprear in purple colour. While Escherichia coli is also in rod-shaped and appear in red colour. These bacteria will cause food poisoning to consumer.

Every food item we eat is biological in origin. The porridge provides a good source of nutrient to bacteria. When bacteria have nutrients (food), moisture, and favorable temperatures, they grow rapidly, increasing in numbers to the point where some types of bacteria can cause illness. Bacteria grow most rapidly in the range of temperatures between 5oC and 60C, some doubling in number in as little as 20 minutes. Some types will produce toxins that are not destroyed by cooking. Food poisoning is mainly caused by different and less widespread bacteria. As they grow, microorganisms release their own enzymes into the liquid surrounding them, and absorb the products of external digestion. This is the main basis of microbial food spoilage, which lowers its nutritional value. Besides, Bacteria may also produce waste products which act as poisons or toxins, thus causing the renowned ill-effects. Since food-poisoning bacteria are often present on many foods, knowing the characteristics of such bacteria is essential to an effective control program. B. cereus is found in dust, soil and spices. It can survive normal cooking as a heat-resistant spore, and normal oxygen atmosphere, then produce a large number of cells if the storage temperature is incorrect. Starchy foods such as rice or porridge, macaroni and potato dishes are most often involved. The spores may be present on raw foods, and their ability to survive high cooking temperatures requires that cooked foods be served hot or cooled rapidly to prevent the growth of this bacteria. Therefore, Improper holding and strorage temperatures after cooking, the food will be spoilt and will cause mild case of diarrhea and some nausea within 12 to 24 hours.

Escherichia coli Can produce toxins that are heat stable and others that are heat-sensitive. The major source of this bacteria in the environment is probably the feces of infected humans, but there may also be animal reservoirs. Feces and untreated water are the most likely sources for contamination of food. Meat and cheeses are involved the growth of this bacteria. It will cause diarrhea and abdominal cramps to consumer.

To preventing food poisoning, food handler need to wash their hand and utensils thoroughly before and after handling raw foods to prevent recontamination of cooked foods. Food consumer also need to wash hand before and after consume food. Next, refrigerated foods must Keep below 40 degrees F while hot foods must serve immediately or keep them heated above 140 degrees F. Besides, canned foods need to heat thoroughly before tasting. If a food has been left in the "Danger Zone" between 5C and 60C for more than 2 hours, discard it, even though it may look and smell good.

Streak plate technique is applied to obtain single colony of a microorganism. From this experiment, we successfully obtain the single colony of all microorganisms. There are Salmonella spp. and Bacillus

spp Staphylococcus aureus and Escherichia coli. The morphology of the colonies can be observe clearly. This is because we used quadrant streak technique to allow sequential dilution of the original microbial material over the entire surface of the fresh agar. Moreover, we also used the entire surface of the plate, not just the middle of the plate and streak slightly so that do not gouge the agar.
Growth of bacterial cultures on agar slants can provide us with useful information concerning motility, pigmentation and oxygen requirements. While there is variation even among individual strains of the same species, some characteristics are distinctive, thus can aid in the beginning steps of identification.

In general, bacterial growth on slants ranges from even (following the line of the original streak), to irregular (slight spreading from the original line), to spreading (the organisms cover the entire surface of the slant). From our results, the growth pattern of Salmonella spp. and Bacillus spp. Are effuse(spreading). Next, the growth pattern of Staphylococcus aureus and Escherichia coli are Echinulate and filiform (thread-like) respectively.

Single colony from the streak dilution plate and agar slant was taken for gram staining to identify the characteristics of the microorganism. The gram staining result shows same characteristics of cell morphology by comparing the actual characteristics of the microorganism. This shows that there is no contamination occurs during streaking on agar plate and agar slant. Conclusion Form this experiment, the bacteria that found in the porridge are Bacillus cereus and Escherichia coli. We get the result by using serial dilution, spread plate method and gram staining. For serial dilution method, increase dilution of sample will decrease the colony count on the agar plate. Next, streaking on agar plate can also get the single colony of bacteria. While streaking on agar slant can roughly identify the bacteria by its motility. References Monroe T. Morgan,(2003), Environmental Health, 3rd edition,Thomson Wadsworth. Tortora, (2001), Microbiology, An Introduction, 7th edition, Benjamin Cummings.
Bacterial Food Poisoning. (n.d.). Retrieved January 8, 2010, from http://aggiehorticulture.tamu.edu/extension/poison.html Food Spoilage And Additives. (n.d.). Retrieved January 9, 2010, from http://www.biotopics.co.uk/pot/foodsp.html

Studentguide.in. (n.d.). Retrieved January 9, 2010, from Microbial Nutrition and Growth: http://www.studentsguide.in/microbiology/microbial-nutrition-growth/growth-characteristics.html MicrobeLibrary.org. (n.d.). Retrieved January 9, 2010, from The Streak Plate Protocol: http://www.microbelibrary.org/edzine/details.asp?id=2808

Growth Characteristics Each bacterium when grows on a medium, shows specific growth appearance or characteristics. These specific characteristics are generally useful in the identification of unknown culture of bacteria. To observe growth characteristic, the bacterial culture is cultivated either on the surface of solid medium (agar plates and agar slope) or in liquid medium (broth). To study the growth of bacteria on agar plate and agar slant following parameters are considered Culture Characteristics of Bacteria

1. Punctiform 4. Rhizoid

2. Granular 5. Filamentous Growth on Agar Slant

3. Circular 6. Irregular

1. Piliform 4. Effuse

2. Echinulate 5. Arboriscent

3. Beaded 6. Rhizoid

1. Size of the colony: small, large, or spreading. 2. Elevation, margin and type of colony on the surface. 3. Pigmentation: pigmented (red, brown, yellow and violet) or nonpigmented. 4. Optical features: opaque, translucent or opalescent. The broth cultures can be examined to find the amount of growth, distribution of growth and

order of growth. The growth can be observed on surface, all over or settled at the bottom. Every bacterial type exhibits peculiar growth characteristics. Thus, analysis of culturing characteristics of bacteria becomes useful in their identification. However, one has to carry out additional tests such as biochemical, morphological and serological to identify the species.

Broths
When bacteria are grown in broths such as trypticase soy broth (TSB), they may exhibit patterns of growth ranging from a sediment at the bottom of the tube, turbid growth throughout the tube, or a pellicle (thick growth at the top of the tube). Pellicle formation is sometimes due to a affinity for oxygen, but may also be the result of hydrophobic compounds present in the cell wall or the general formation of dry, light colonies. Also, if an organism produces and releases soluble pigments, these will spread into the broth and change its color. Here are two examples of growth patterns in broth after 48 hours incubation at 37o C:

This broth contains the acid-fast species Mycobacterium smegmatis. Note the pellicle on the surface of the broth which forms due to the high concentration of hydrophobic mycolic acids embedded in the cell wall of this species.

This broth contains Serratia marcescens, a gram-negative rod. Observe the turbid appearence of the broth and the red color present in both the sediment and pellicle, which is the result of the nonsoluble pigment prodigiosin produced by this bacterium.