Skills to be practised in diagnostic microbiology

Skills to be practised in diagnostic microbiology

1. WHY MICROBIOLOGISTS TO BE SKILLED PROFESSIONALS?
Dr.T.V.Rao MD
The words of our teachers who were dedicated to the profession is to be
remembered and practised, When I was observing the reporting by my Teacher Dr Joga Lakshmi a
retired Emeritus Professor of Microbiology at Andhra Medical College, Visakhapatnam of the
routine Bacteriological cultures I told Madam I am not able to adopt well with the separation of
Mixture of bacteria she just told the true worth of MD in Microbiology is identifying a pathogen from
non-pathogenic Microbes, No one can become a competent Microbiologist unless we have power of
discrimination from commensals to Infectious bacteria, Wisdom prevailed in me to practice with
dedication to separate the mixtures, it helped me in many ways to teach many younger generation
of Microbiologists, We had few Media, only available medium was Nutrient agar, Mac Conkey agar
with few available Blood Agar . To get few sets pf IMVIC tubes was more than difficult with limited
resources in a Government medical College, I learned many matters from our technicians as I used
to see when the Technicians were streaking the culture plates, single colonies can be identified and
separated by using the streak plate technique this is true skill in separating mixture of bacteria
Today many neglect the bench work, and many younger generation of Microbiologists think it is the
duty of technicians they are only for reporting the culture isolates, My real Guru continues to Late Dr
CSV Subramanianm Chief of Laboratory services Apollo Hospital Madras(1992} advised me to be
good bench worker you will become a skilled Microbiologist or else Laboratory Technicians will
overtake you one day, I am happy I sustained in the Profession with bench work and practice of
Microbiology with Additional methods used to physically separate bacteria include; dilution in
liquid, the pour plate where diluted cultures are poured onto a solid medium spreading over its
surface and the spread plate or where diluted cultures are spread onto a solid medium and thus
distributed over the medium surface).
Because the task of isolating a given bacterial species from a mixture can often be simplified by
taking advantage of a particular nutritional requirement of a given species, constituents of media
can be varied to select for, or differentiate among bacterial types. Media designed for selection and
differentiation are described below.
Selective media will permit the growth of one type of bacteria while preventing the growth of other
types. However we had few when I was a Postgraduate Students however with raise of Hi Media
we have plenty of media, as This will facilitate the isolation of a desired species. MacConkey’s
medium is selective. It contains bile salts and crystal violet, these will inhibit the growth of Gram-
positive organisms. Other examples include phenylethyl alcohol agar, which inhibits or slows the
growth of Gram-negative organisms, and mannitol salt agar which inhibits the growth of salt
intolerant organisms.
All the post graduate should be proficient with Differential media which will allow visual
differentiation between two or more species of bacteria. Examples include blood agar, MacConkey’s
medium and Mannitol Salt agar. Colonies growing on blood agar are differentiated by haemolysis
patterns (greening - alpha haemolysis, clearing - beta haemolysis and no haemolysis gamma).
Using MacConkey’s medium, lactose fermenting and lactose non-fermenting bacteria can be
distinguished. Organisms that are able to ferment lactose will produce an acid end-product that
causes a change in the pH of the surrounding media. A pH indicator (neutral red), is present in the
media and changes from yellow to red in an acidic environment. Lactose fermenting bacteria
growing on

2. MacConkey’s media will appear pink whereas non-lactose fermenters will be white. I practically did
my thesis with few media as Mac conkey agar, DCA Nutrient agar, Few other organisms identified
with Mannitol salt agar allows discrimination among salt tolerant organisms. Those that ferment
mannitol will produce acid turning the pH indicator (phenol red) to a yellow colour. Those that
cannot ferment mannitol will leave the media the red colour of phenol red at neutral ph.
USE THE HAND SKILLS AND BE SYSTEMATIC IN APPROACH
To test for the growth of numerous bacterial species on one petri dish, the plate may be streaked in
sectors. The streaking will not allow observation of isolated colonies, but will allow the
determination of positive or negative growth. On the bottom of the petri dish, use a marker to
divide each plate into sectors: e.g. three sectors for three bacterial test species.
Inoculate by streaking each sector with test culture. Streak each sector carefully, staying within the
sector margins to minimize the possibility of cross contamination.
Incubate plate in an inverted position and wrapped in a plastic bag. For most cultures incubation at
37oC for 24 hours is sufficient to allow growth.
What new information about your organisms have you gained from this lab? PUT IN CLEAR WORDS
Can you write sentences like this? As child learning Alphabets
“Isolate A is a salt tolerant, alpha haemolytic, Gram positive cocci. It grows best in the mesophilic
range and is lactose fermenting.”?
Did the MacConky plate data agree with Gram stain results? (If not, repeat your Gram stain!)
With new information gained in this lab, can you distinguish among your isolates?
A beginning of good automation and Molecular biology is based on identifying the true pathogen
from many contaminations we face in our laboratories
I wish to state there is no rapid diagnostic than Gram stain if we pay little attention who can DO
many wondrous IN diagnosis in many trying circumstances
NEVER FORGET TRUE MICROBIOLOGY IS DEMOSTARING OUR SKILLS RATHER THAN TALKING
COMPLEX WORDS AS MANY LEARNED DO TODAY?
AN OUNCE OF PRACTISE MAKES BETTER MICROBIOLOGITS FOR THE FUTURE THAN TONS OF
DISCUSSIONS AS IT HAPPENS IN POST GRADUATE INSTITUTES
ALL QUALFIED MEDICAL MICROBIOLOGITS LOOK FORWARD TO SEE YOUR SKILLS AND TALENTS
IMPROVING RATHER THAN YOUR AUTHORITY OF POSTION IN HIERARCHY
Dr.T.V.Rao MD Professor of Microbiology Freelance writer