WHY R-CARDS AND OTHER MEDIA REQUIRE DIFFERENT INCUBATION TIMES AND TEMPERATURES FOR USE IN ACQUIRING SATISFACTORY RESULTS FOR THE USER
THE GROWTH CURVE FOR MICROBES
When a tube of sterile Nutrient Broth has a drop (or less) of material containing hundreds (or more) of living microbes, there is a shock to those microbes that may result in an initial reduction of viable cells (“Lag phase”).
Then when the microbes adjust and use the nutrients in the broth, they begin to reproduce rapidly. The initial clear broth turns cloudy from the masses of healthy microbes growing in it (Log Phase). These microbes are reproducing rapidly with very little death because they have a large supply of nutrients and a very low concentration of toxic waste products. This vigorous new culture contains a minimum of dying, mutating cells and is the ideal population to use in experiments. It should be noted that different genera and species of bacteria are inherently different in their growth rates. For example, most species of Escherichia coli grow very rapidly and may double their population in as little as 10 minutes in ideal conditions, while other bacterial species may take longer than an hour to double in ideal conditions. (This explains why some experimental results can be read and used very quickly and others may take 10 or more times longer.)
As the broth culture ages, it reaches the Stationary Phase, where there may be equal numbers of bacteria dying as those reproducing. This is largely due to the increased concentration of toxic waste products accumulating. At this point, when the culture is used for experimentation, some of the bacteria are no longer in good condition, and will take longer to recover and reproduce. This results in a difference between the newly reproduced bacteria and the ones which are dying.
It is therefore logical as more and more bacteria are declining and entering the Death Phase, they are not as healthy and reproduce as fast as those that are newly being reproductive. It takes time for these near death to recover and grow into new colonies.
Taking the previous information into account, it is logical that young, vigorous bacteria will grow faster and produce colonies that are visible to the experimenter than colonies that grow from an original almost dead microbe.
This is why some experimenters are satisfied with what may be described as a PRESENSE/ABSENSE reading of results without checking their cultures at a later incubation time to see if additional Colonies have developed from bacteria of the target species that were in the Death Phase and took longer to recover and produce visible Colonies.
THE PRECEEDING INFORMATION IS MEANT TO EXPLAIN WHY THE PROTOCOLS FOR THE VARIOUS METHODS FOR VAVIOUS MICROBE SPECIES COMMONLY LISTS A TIME RANGE FOR EACH DIFFERENT SPECIES. FOR EXAMPLE, WE RECOMMEND THAT FOR E. COLI (A FAST GROWING BACTERIUM) COLONIES FROM YOUNG, HEALTHY CELLS CAN BE COUNTED AS EARLY AS 14-16 HOURS. HOWEVER THERE MAY BE COLONIES FROM OLD (NEAR DEATH) CELLS APPEARING UP TO 24 HOURS. (HOWEVER, WE RECOMMEND THAT IF ANY NEW COLONIES APPEAR BEYOND 24 HOURS, THEY SHOULD NOT BE COUNTED AS E. COLI WITHOUT FURTHER TESTING.)
HOWEVER, SOME STRAINS OF ENTEROCOCCUS FAECALIS.A DIFFERENT FECAL SPECIES, MAY ALSO SHOW EARLY, BUT DEATH PHASE CELLS MAY PERSIST LONGER, SO OUR RECOMMENDATION FOR IT IS TO COUNT COLONIES THAT GROW AS LATE AS 40 HOURS INCUBATION.
MICROBIOLOGY IS A FIELD OF SCIENCE THAT INCLUDES THE ART AND BEAUTY OF OUR WORLD, AND IT TOUCHES EVERYONE. LIFE AS WE KNOW IT COULD NOT EXIST WITHOUT MICROORGANISMS. MOST MICROBES ARE BENEFICIAL, BUT SOME CAUSE ILLNESS AND DEATH IF NOT CONTROLLED. THEY CONTRIBUTE GREATLY TO A WORLD FULL OF SURPRISES. A GOOD MOTTO FOR THE MICROBIOLOGIST IS “THE EXCEPTION IS THE RULE!!”
JONATHAN N. ROTH, PH.D. & C.E.O.
ROTH BIOSCIENCE, LLC
OCTOBER 5, 2022