Angeles University Foundation Angeles City, Philippines A.Y.

2013 2014

College of Allied Medical Professions Bachelor of Science in Medical Technology

Final Written Report (FINALS) Bacteriologic Assay of Drinking Water

Submitted by: Group 5 BSMT3E Eure, Verna L. Jocson, Jancy R. Malinit, Agatha L. Submitted to: Ms. Sandra Kathrina Carlos, RMT Mrs. Mary Kimberly Pongco, RMT Dr. Maria Cielo Asuncion

I. Introduction
Water is a necessity for all living things to live. Humans in particular are most responsible for proper sanitation and maintenance of water sources. Despite the precautionary measures taken for water treatment, there are continuous instances wherein contamination may occur. Ingestion of contaminated water could be life-threatening for both humans and animals; this is why it is important to conduct water analysis. Infectious diseases may be transmitted by several routes, and one of them is water. It is possible that water could be contaminated as a result of improper sanitary measures. Such contaminants include feces, chemical contaminants, parasites such as Plasmodium falciparum and Naegleria fowleri - causative agent of malaria and causative agent of brain-eating amoeba respectively. Common microorganisms associated with water contamination are Shigella, Salmonella, enteropathogenic Escherichia coli, and hepatitis A virus (HAV). Even after water has been treated, if it is not re-examined for confirmation of decontamination, it can still harbor disease-causing microorganisms. Escherichia coli in particular are a very common water contaminant. E.coli is capable of causing diarrhea among adults and infants. Infants in particular are more affected, considering that their immunity has not been fully developed. Since the bodys water is lost during diarrhea, dehydration can be the cause of death. E.coli diarrhea is also known as travelers diarrhea because it is often contracted by tourists that travel to tropical countries like the Philippines, and Montezumas Revenge which was coined from the name of an Aztec chief killed at the hands of the Spanish explorer, Cortez. E.coli diarrhea depends on which virulence factors of the strain it possesses. Travelers diarrhea is caused by Enterotoxigenic Escherichia coli (ETEC) which inhibits the reabsorption of sodium and chloride electrolytes and stimulates secretion of chloride and bicarbonate into the intestinal lumen resulting in water and electrolyte loss which produces severe watery diarrhea. Enterohemorrhagic Escherichia coli (EHEC) secrete Shigella-like toxin verotoxin which has the same mode of action with Shigella. The diarrhea is bloody and the patient experiences severe abdominal cramps. This condition is called Hemorrhagic colitis. EHEC strain Escherichia coli 0157:H7 is associated with Hemolytic uremic syndrome (HUS). Enteroinvasive Escherichia coli (EIEC) also produce small amounts of verotoxin. Verotoxin invades the epithelial cells of the intestine and when the host attempts to get rid of the bacteria, an immune-mediated inflammatory reaction with fever occurs. Escherichia coli are also known to cause Urinary Tract Infection, commonly among women, neonatal meningitis, gram-negative sepsis, and hospital-acquired pneumonia.

II. Methods and Materials I. Materials and Procedure

In this experiment the needed materials includes: o o o o Alcohol Lamp Test tube rack Inoculating loop Serological pipettes (sterilized) o o o Specimen containers (sterilized) Culture media- presumptive, confirmed, completed test Gram staining set.

A. Sample Collection 1. Tap water should be free from any attachment. Wipe the outlet with clean cloth to remove any dirt and contamination. 2. Let the outlet flow for 1-2 minutes at a maximum rate then turn it off. Using flame from an alcohol lamp or cigarette lighter. Heat the opening of the outlet. 3. Turn it on again slowly. And allow water to flow for 1-2 at a medium rate. Hold the bottle under the faucet to fill. Leave some space. This will allow shaking of water prior to analysis. Cover the container with light-protecting material. B. Specimen processing A. Presumptive test (Multiple Fermentation Tube Technique-Lactose broth) i. Shake or mix the water sample to have a equal dispersion of bacteria. ii. Open the bottle. 5 ml of the sample is to be pipettedd using a sterile pipette into each five tubes containing 5 ml presumptive lactose broth. iii. Shake tubes carefully to allow spreading of sample uniformly into the medium. iv. Incubate at 35 C =+ .5 degree Celsius for 2 hrs. vi. at the end of the incubation period, check for the presence of gas for each tube. Gas is seen inside the Durham tube or tiny bubble upon light shaking is recorded as positive. If the test is negative, there is no presence of bubbles reincubate tube for 2 hours. v. record result. State as positive if there I the presence of bubbles regardless of the amount. B. Confirmed test (Multiple Fermentation Tube Technique Brilliant Green Bile Lactose broth) i. this test is should be carried out at the end of a 24-hour and 48-hour incubation.

ii. using a sterile loop, transfer a loopful of each of the primary fermentation tube that ho the gas formation into the BGBL. iii. After incubation, examine the gas formation. If none, gently shake the tube and observed for the effervescence iv. Negative tube are subjected to re incubation v. record result Positive Tubes 0 1 2 3 4 5 MPN/100 ml <2.2 2.2 5.1 9.2 16 >16

C. Completed Test i. examine growth o EMB (Black colonies with green metallic sheen) ii. Perform the IMViC Test and Gram Stain to confirm the presence of the indicator strain iii. Interpret results

III. Results & Findings Test Sample Turbidity and Gas Production Control Turbidity and Gas Production

Presumptive

*Positive for Gas Production in Brilliant Green Bile Lactose Broth *Positive Growth with Green Metallic Sheen

*Positive for Gas Production in Brilliant Green Bile Lactose Broth *Positive Growth with Green Metallic Sheen

Confirmed

*Gram (-) rods in singles, pairs and clusters

*Gram (-) rods in singles, pairs and clusters *Indole Positive *Citrate Positive

Completed

**Expected Results for: Methyl Red Vogues Proskauer Citrate Utilization VISIBLE Green Metallic Sheen

a. Methyl Red (+) b. Vogues Proskauer (-)

IV. Summary and Discussion Water analysis consists of three parts, the presumptive test which uses lactose broth, a classical medium used in the presumptive testing for coliforms and for the enrichment of Salmonella. The experiment was conducted by using water from the Medical Technology supply room as the test sample and Escherichia coli as the control sample. Both the test and control sample exhibited positive results which were indicated by the presence of effervescences and gas production in the lactose broth. Having positive results in the presumptive test, we need to further confirm them and that is the second part of the analysis. For the confirmed test we use Brilliant Green Bile Lactose Broth which is a selective enrichment and enumeration by titer determination or MPN method of E. coli and other fecal coliforms from water, milk, food and other material. The results for the experimentation are both positive for the sample and control, indicated by the gas production in the Durham tube and presence of effervescence. The last part is the complete test which consists of interpretation of growth on EMB, Gram staining and IMViC. The sample and control were subjected to gram staining and they both stained red, classifying them as Gram negative. For IMViC, the result of the Indole test for the control is positive which correlates with the expected positive result. Being unable to finish the IMViC test, the expected results shall be discussed for the Methyl Red and Vogues Proskauer. The control sample is expected to be positive in Methyl Red and negative in Voges Proskauer. The citrate utilization test differentiates organisms that can utilize citrate as their sole carbohydrate source. Results show that the control sample tested positive for citrate test, which deviates from the expected result to negative for citrate.

Questions for Research 1. Define the term coliform. What is its significance in water bacteriology? Coliform is defined as rod-shaped gram negative non-spore forming bacteria which can ferment with the production of acid and gas when incubated at 35 to 370C. They are important in water bacteriology because they are used as water quality indicators. They may be associated with the sources of pathogens contaminating water. The analysis of drinking water for coliforms is relatively simple, economical and efficient. 2. What circumstances might be responsible for a positive presumptive test on a sample which, on subsequent, does not yield a positive confirmed test? Presumptive test shows gas formation in the Durham tube. However, gas formation may be due to non-coliform bacteria so we need to confirm it by using confirmed tests. If it does not yield a positive confirmed test, it is possible that it is not a coliform bacteria such as Clostridium.

3. Discuss some indicators of fecal pollution. It is unusual to find fecal indicator bacteria and even some pathogens in natural environments. Giardia lamblia which is a protozoan is an example. Another pathogen associated with fecal pollution includes bacteria such as Pseudomonas and Helicobacter and others are viruses. 4. Give at least two other methods for bacteriologic analysis of water and discuss the principle and modifications employed by each. Method 1: ATP Testing ATP testing is a process of rapidly measuring microorganisms in water through detection of ATP. ATP is a molecule found only in and around living cells and as such it gives a direct measure of biological concentration and health. ATP is quantified by measuring the light produced through its reaction with the naturally occurring enzymes firefly luciferase using a lucinometer. The amount of light produced is directly proportional to the amount of biological energy in the sample. Method 2: Membrane Filtration Most modern laboratories use a refinement of total plate count in which serial dilutions of the sample are vacuum filtered through purpose made membrane filters and these filters are themselves laid on nutrient medium with sealed plates. The methodology is otherwise similar to conventional total plate counts. Membranes have a printed millimeter grid printed on and can be reliably used to count the number of colonies under a binocular microscope.

V. Journal VI. References http://www.microbelibrary.org/component/resource/laboratory-test/2742-methyl-redvoges-proskauer-tests http://www.microbelibrary.org/component/resource/laboratory-test/2757-citrate-test http://web.cn.edu/stkarr/physiolo.htm http://www.sanitationtools.com/Category.asp?Category=64 http://www.sigmaaldrich.com/catalog/product/fluka/94792?lang=en&region=PH Gladwin, Mark M.D., Trattler, Bill M.D., Clinical Microbiology Made Ridiculously Simple Delost, Maria Dannessa, Introduction to Diagnostic Microbiology: A Text and Workbook