Hi! Working on microbiological methods for an undergraduate thesis project. Wondering how detailed we have to be identifying the bacterial isolates we have in order to have respectable and meaningful data.
We aim to identify potentially pathogenic bacteria from holy water fonts in churches. We'll do so by collecting holy water samples from the fonts, then inoculate them into selective media.
We're focusing on Streptococcus and Staphylococcus-- so we're inoculating the diluted samples into Mannitol Salt Agar (selects for Staph) and (selects for Strep). So with these media we narrow down the bacteria to the genus level. For a meaningful study, do we have to identify all colonies up to the species level?
Initially we planned to do this via DNA sequencing of colonies, but it proved too costly. What biochemical tests should we focus on to differentiate the Strep species and the Staph species, while reducing costs/redundancies?
We're thinking of Catalase Test, CAMP test, Taxos A and P tests for Strep; and Blood Agar plating, Coagulase Test, and Catalase Test for Staph. Would these be enough to identify the species under Streptococcus and Staphylococcus? Other suggestions of other tests that would simplify the identification would be much appreciated.
Thank you very much!
For Staph and strepto a wise choice is to use API
API Gram positive Identification
- API Staph – Overnight identification of clinical staphylococci and micrococci
- RAPIDEC® Staph – 2-hour identification of the commonly occurring staphylococci
- API 20 Strep – 4 or 24-hour identification of streptococci and enterococci
- http://www.biomerieux-usa.com/clinical/api
For Staph and strepto a wise choice is to use API
API Gram positive Identification
- API Staph – Overnight identification of clinical staphylococci and micrococci
- RAPIDEC® Staph – 2-hour identification of the commonly occurring staphylococci
- API 20 Strep – 4 or 24-hour identification of streptococci and enterococci
- http://www.biomerieux-usa.com/clinical/api
Catalase test, coagulase test and hemolysis test should give you a better differentiation of Staph and Strep. All the best.
As you apparently have limited resources - to expand on Pasanth's good suggestions, Gram stain, hemolysis(sheep blood agar), catalase and coagulase for Staphylococcus aureus and Gram stain, hemolysis, catalase and bacitracin inhibition for Gp. A strep (Streptococcus pyogenes). You can find appropriate reactions described in the internet.
You should also review the literature (google scholar). there's a fair amount of info on microbes recovered from holy water. e.g.Article Holy water fonts are reservoirs of pathogenic bacteria
Thank you very much for your answers! We will focus on Catalase test, Coagulase test and Hemolysis test.
If you grow up your bacteria on sheep blood agar plates, the staphylococcal colonies will be larger than streptococcal colonies and most of the pathogenic bacteria will be beta hemolytic (a clear zone around the colonies). Confirm which they are by Gram staining (microscopic arrangement of streptococci are chains while staphylococci are in clusters). Streptococci are catalase negative while staphylococci are positive, but you can't perform catalase test from blood agar, since red blood cells are loaded with catalase.
If you use only MSA:
. In MSA you will not have any growth of Gram negative and Streptococcus. High salt concentration inhibit growth. So you must choose other medium for Streptococcus.
With MSA, the yellow colonies are mostly Staphylococcus Coag+ (as S. aureus). Staph coag negative are pink or red. You can confirm with complementary test as catalse (cat+), gram and the coagulase test.
For Streptococcus you must inoculate a pre-warm (37ºC) Sheep Blood Agar or a TSA with 5% of Blood Sheep. Incubate and pick colonies for TSA medium and realize test of catalase (negative), gram (positive cocci).
beta hemolytic colonies must be tested with bacitracin. Bacitracin sensitive are S.pyogenes. Bacitracin Resistant are other beta hemolytic Streptococci. (use APIStrep for identification).
alfa hemolytic colonies must be tested with optochin. Optochin sensitive are S.pneumoniae. Optochin Resistant are other alfa hemolytic Streptococci. (use APIStrep for identification)
Non-hemolitic colonies rarely cause disease. (use APIStrep for identification)
In the first place, why restrict yourself to only two bacteria. I suggest you carry out a holistic pathogenic bacterial assay on the font water. The result will be more appreciated and helpful in public health matters.
In the first place: why restrict yourself to only two bacteria. I suggest you carry out a holistic pathogenic bacterial assay on the font water. The result will be more appreciated and helpful in public health matters.
In the second place: If I were prefer to isolate two types of bacteria Staph & Strep. You should do the following tests (Gram stain, hemolysis (sheep blood agar), catalase and coagulase).
I suggest that you also try to isolate gram-negative bacilli and detect multiresistant in water
Hi, as your test sample is holy water, I would prefer to test fecal coliform and other pathogenic E. coli. as the first attempt. Thank you.
For easily and rapidly diagnose, you can use API test and VITEK 2 system ... good luck.
Hi there, Really, it is hard to limit as there are possibilities of wide range of waterborne microbial pathogens, asbacteria, viruses, protozoa and helminths, etc. . You better confine your test to common waterborne pathogenic bacteria, such as E. coli O157:H7, Salmonella, Salmonella typhi, Shigella, Campylobacter, Vibrio cholerae, Pseudomonas etc. If your time and fund permit, I suggest to make a survey of the conditions of the water resevoirs too. Good luck.
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