Water Bacteriology Flashcards
What is Water Microbiology
- deals with the study of microorganisms and parasites in water
- here in Phil, institutions are analyzing bacteria (limited to) in water only
- ensure safe consumption of water
Sample used in water microbiology
- Water
not body fulids
3 sections of Water testing laboratories
Environmental laboratory
- for metal analysis in water
Wet laboratory
- for the physical and chemical analysis of water
Microbiological Laboratory
- for bacteriological analysis if water
3 types of water dealt with in a water microbiology lab
- drinking water
- waste water
- recreational water
Dechlorinating agent used in water microbiology
Sodium Thiosulfate
Who are the analyst required to work in water microbiology Lab
registered: in DENR
- medical technologist
- microbiologist
- food technologist
In DOH they accept:
- chemist
- biologist
must undergo special training by East avenue medical center
Quality of a good drinking water
- clear
- colorless
- free from objectionable taste and odor
- Must not contain any substance or organisms, chemicals, and radioactive materials
Why and when to detect microorganism in water
❑ Outbreak investigation of waterborne diseases
❑ Assesing safety, stability of water and water products for public consumption
❑ Determining level of sanitation during product preparation (e.g., in restaurants)
❑ Regulatory compliance (in business)
❑ Incidence surveys
True or False:
In water microbiology specific isolation of the bacteria is necessary
False
- the test only limits to identifying the preence and abscence of Coliform bacteria
are microorganisms such as bacteria and viruses in water bodies, which are utilized as a surrogate to evaluate the presence of pathogens in that environment.
- Indicator organisms or are the COLIFORM bacteria
The most common Coliform (facultative anaerobe) bacteria
- Escherichia coli (most numerous)
- Enterobacter
- Klebsiella
- Citrobacter
True or False:
If the water tested positive with the indicator bactera, it mean that there is also possible presence of all relevant pathogens?
- True (almost 90%)
2 classifications of Coliforms (facultative anaerobe)
- Total Coliform (found everywhere)
- Fecal/ Thermotolerant Coliform (found in fecal material)
Characteristic of Coliforms
❑Facultative anerobe ❑ Gram negative bacilli ❑ Non-spore forming ❑ Ferment lactose at 35oC for 48 hours - (total choliforms) 44.5oC for 24 hours- (Fecal coliforms) ❑ Non- pathogenic
Bacterial detection Methods
❑ DIRECT ENUMERATION
❑ INDIRECT ENUMERATION
❑ PATHOGEN ISOLATION AND IDENTIFICATION
Direct Enumeration can be done through?
❑ Microscopic Count
❑ CFU count using non-selective media
Indirect Enumeration can be done through?
❑ Most Probable Number (MPN) or Multiple Tube Fermentation Technique
- quantitative estimation
- applied in 3 types of water
Pathogen Isolation and Identification can be done through
❑ Series of biochemical tests
Acceptable water testing method mandated by Philippine National Standards for Drinking Water. Department of Health. 2017.
❑ Multiple Tube Fermentation Technique
❑ Standard Membrane Filtration Technique
❑ Heterotrophic Plate Count (Pour or Spread Plate)
(the above are most common)
❑ Enzyme Substrate Coliform Test
❑ Delayed Incubation Total Coliform Membrane Filter Technique
❑ Simultaneous Detection of Total Coliform and and E.coli by
Dual Chromogen Membrane Filter Technique
Standard Membrane Filtration Technique
and Heterotrophic Plate Count (Pour or Spread Plate) is applied in what type of water
- drinking water
waste water can be done on these methods however, the density of bacteria limits the method.
technique that can be used to classify organisms in all of the 3 types of water
Multiple Tube Fermentation Technique
This is where reglations and rules are made or where the submission of requirements in water testing is passed
Philippine National Standards for Drinking Water. Department of Health.
Storage of Culture Media in Water microbiology laboratories
Store dehydrated media (powder/granulated) in:
- tightly closed bottles
- in the dark
- <30 degrees celcius
- Low humidity (<50%)
True or False:
If culture medium can still be used even if they become discolored or become
caked and lose the character of a free flowing powder.
- False
do not use them
Sterilization of culture media tubes is done through
-Autoclaving at 121 degrees celcius C, 15 psi for 15 minutes.
Note: Dispense first before autoclaving
Record final pH after autoclaving
True or False:
Culture media used in Membrane filtration technique can be autoclaved
- False
some of their components are carcinogenic are tends to deteriorate
Water used in preparation of culture medias in water microbiology lab
- Distilled or deminaralized water
True or False:
Fermentation tubes can be store in 25 °C for more than 2 weeks.
False
- because evaporation may
proceed rapidly under these conditions, resulting in marked changes in concentration of the ingredients,
What must de done in liquid media fermentation or carbohydrate tubes if they are refrigerated?
- bring in room temperature and discard tubes that contain bubbles
What are the labels necessary to be included in the culture media?
❑ Media/Reagent Name
❑ Date prepared
❑ Expiration date
❑ Prepared by
Preparation of Culture media plates
- Autoclave first then dispense
- 4 to 6 mL into 50 mm x 9 mm petri dishes is used
- solidify at room temp.
- store in refrigerator at inverted position in a plastic bag or tight container
Note: prefreably use for no longer than one week
In Multiple Fermentation technique:
Presumptive identification is done in what culture media
Lauryl Sulfate Broth
or Lauryl Tryptose Broth (LSB/LTB)
- for both total and fecal coliforms
In Multiple Fermentation technique:
Confirmatory identification is done in what culture media
Brilliant Green Lactose Broth (BGLB)
- For Total Coliforms
detection
EC (Escherichia coli) Broth
- For Fecal Coliforms detection
The accepted dilution water used in Multiple tube fermentation
❑ 0.1 % Peptone water
❑ Stock phosphate buffer solution
❑ Magnesium chloride stock solution
True or False:
It is not advisable to suspend a sample in any dilution water for more thab 30 mins at RT.
- True
since injury, death, or multiplication may occur
Principle behind Multiple tube fermentation technique
- Serial Dilution
If 99 ± 2.0 mL solution is needed what is the amount of water to be dispensed
100 mL
If 9 ± 0.2 mL mL solution is needed what is the amount of water to be dispensed
10 mL
How is 0.1 % peptone water prepared?
1 gram of peptone water and 1000 mL of distilled water
In MEMBRANE FILTRATION TECHNIQUE:
What agar is used for detection of Total coliforms
- ENDO agar
95% ethanol is added
In MEMBRANE FILTRATION TECHNIQUE:
What agar is used for detection of Fecal coliforms
- Fc agar
Sodium hydroxide (0.2N NaOH) and 1% Rosolic acid is added
What is added to Fc agar?
Sodium hydroxide (0.2N NaOH) and 1% Rosolic acid
In MEMBRANE FILTRATION TECHNIQUE:
What agars are used for Escherichia coli detection
- Urea broth
- mTEC agar
Inhibitory agents of Endo agar
- basic fuschin and soldium sulfide
inhibits gram (+) bacteria and bacteria that are not total coliform
Positive result on Endo agar medium
- Pink colonies/ lactose fermenters
differentiates bacteria on the basis of lactose fermentation
Inhibitory agents of Fc Agar
- bile salts
The most common culture medium used in Heterotrophic plate count technique
Plate Count agar - Pour plate or Spread plate method
What is the difference between the Pour plate and Spread plate method
- in pour plate the agar is poured on the time of analysis whereas on spread plate method you have to prepare the agar first before analysis.
For wastewater and recreational water, what is added on the collection bottle?
2-3 drops 10% sodium thiosulfate —> then autoclave
For drinking water, what is added on the collection bottle?
2-3 drops of 3% Sodium thiosulfate —> then autoclave
For how many minutes will you run the water, when collecting samples in faucets or water dispenser
2-3 minutes —> heat the mouth or apply sodium hypochlorite —> run again 2-3 mins—> collect sample
Positive control for Total Coliforms
Escherichia coli
Enterobacter aerogenes
Negative control for Total coliforms
Staphylococcus aureus
Pseudomonas spp.
Positive control for Fecal Coliforms
Escherichia coli
Negative control for Fecal coliforms
Enterobacter aerogenes
Streptococcus faecalis
How to check purity of culture organism (if it’s pure culture)
- Gram staining or thru Eosin methylene blue (EMB agar)
What do E.coli looks like in Eosin methylene blu medium
- greenish-metallic chain
what should be done in every batch analysis of the same procedure to know the efficiency of the culture media and reagents prepared?
Blank test
What agar is used for Air Micro quality check?
- Plate count agar
