You have just survived your \"Welcome to the Enumeration of Bacteria Thunderdome

Question

You have just survived your "Welcome to the Enumeration of Bacteria Thunderdome" Experience in General Microbiology Lab when you happen to come across a neon orange puddle outside of the Science Building. Seeing that it is Friday night and you love science, you decide that you would like to use your spare time to work through all the ways you could enumerate the bacteria present in the neon puddle. You collect 1 liter (L) of the puddle sample to work from. ccount ; orana SS-7 RNA from the puddie. Acridine orange is a nucleic You begin with creating an acid binding dye that emits green fluorescence when bound to double-stranded DNA (ds DNA) and a reddish orange fluorescence when bound to single-stranded DNA or more importantly, RNA. RNA can be detected from samples as an indication of activity and thus viability of the cells being sampled. e from a sa e you co You find that your puddle sample AODC slides appear to have a high amount of green fluorescence and a lower level of reddish orange fluorescence. In fact, you count in one field (terminology for the view of the slide you see through the microscope at that specific position on the slide) and note that there are four times as many green ce are orange cells. What does this tell you about your sample in terms of nudleic acids and potential viability What typeof microscopy are you using? (Typed answer worth 2 points) DNA RNA Following your assessment of the e in name puddle by AODC, you move onto an MPN estimation of the level of coliforms dle sample. You begin by running a preliminary presumptive test and inoculate 5 DSLB with 10mL of your puddle sample, 5 SSLB with 1ml, and 5 SSLB with 0.1mL of the puddle sample. After 48 hours of incubation at 35 C, you find an MPN value of5-1-2What is the MPN value? What does this MPN value mean in terms of your sample enumeration? (Typed answer worth 2 points) (#of What are we counting ey volume 8-11 5-12 63 52-1 DON'T FORGET THE BACK PAGE

Explanation / Answer

In terms of nucleic acid, the sample contains higher concentration of DNA compared to the concentration of RNA. With respect to the viability based on AO staining,the results need to be validated better. Both type of cells, viable and non-viable, will stain green or red based on their nucleic acid content as the dye is cell wall permeable. When it intercalates with DNA, the cells fluoresces green and interaction with the phosphate group of the RNA gives a red fluorescence. Cells that are actively dividing contains a higher concentration of RNA compared to the inactive cells that has a higher concentration of DNA. Based on this, it can be inferred only a lesser number of micro-organisms are active and thus viable. The type of microscopy used is fluorescence microscopy.

The MPN value is 63. This means that 63 coliforms are present per 100ml of the sample. As the collected sample was 1L (1000mL), it can be enumerated that 630 (63*10) coliforms would be present in the collected sample from the puddle.

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