MKBN REPORT
Student number: 38122189
Name: Chaidene Fredericks
ABSTRACT
Antibiotic susceptibility testing and selective and differential media: a
comprehensive overview
Antibiotic susceptibility testing specifies effective dosage measures for the proper
management of antibiotic susceptibility as a means to control the development of antibiotic
resistance leading to clinical treatment failure (Bhagaban et al. 2019). This technique aims to
test the efficiency of antibiotics on bacterial strains in order to investigate local resistance
patterns. The second technique outlined within this experiment is known as Selective and
differential media which are two types of growth media that provide useful tools for
isolating, identifying, and characterizing microorganisms. Differential media distinguishes
microorganisms and allows for the characterization of microorganisms while selective media
inhibits specific from. Selective and differential media techniques aim to investigate the best
media for the isolation and growth of bacteria to obtain reliable counts and results. There
are various methodologies used to complete both antibiotic susceptibility and selective and
differential media techniques however results outlined only account for the disk diffusion
test and making simple inoculations to study the effects of common cultures on different
media. Experimental results obtained for antibiotic susceptibility uses diameters of
inhibition to determine resistance, susceptibility, or intermediate resistance. Selective and
differential media results are based on the media’s selective and differential properties.
Typically we can view that certain bacteria have grown on media while others have not
grown at all. Evidently, the experimental results obtained support existing data which
concludes a successful experimental procedure.
Introduction
Clinical microbiology laboratories play crucial roles in conducting antimicrobial susceptibility
testing to verify the susceptibility of chosen antimicrobial agents and identify resistance in
individual bacterial isolates (Weinstein et al. 2009). This Empirical technique assesses the
efficiency of antibiotics against pathogens by performing continued susceptibility testing on
individual isolates for species that are known to possess resistance mechanisms. Various
culture comparisons were used for antibiotic susceptibility testing (via Kirby-Bauer disc
diffusion) and selective and differential media to obtain experimental results. The cultures
as listed as follows; Escherichia coli: better known as E.coli which is commonly known to
cause UTI infections, Bacillus subtilis: which is known to cause salmonellosis,
Staphylococcus aureus: the leading cause of skin and soft tissue infections such as abscesses
(S. Chacko 2022) as well as Staphylococcus epidermis: which ranks the most common
species of bacteria to cause infective endocarditis.
The antibiotics used during the experiment to test the susceptibility of the above-mentioned
bacteria are:
1. Amoxicillin- a broad-spectrum antibiotic effective against a wide range of gram-
positive and gram-negative bacteria. It belongs to the beta-lactam class of antibiotics
by inhibiting cell wall synthesis. It binds to penicillin-binding proteins which leads to
the disruption of bacterial cell wall formation and subsequent death.
2. Penicillin- is a narrow-spectrum antibiotic primarily effective against gram-positive
bacteria. It inhibits cell wall synthesis by binding to penicillin-binding proteins. This
disrupts cross-linking of peptidoglycan, leading to cell wall instability and bacterial
lysis
3. Sulfamethoxazole- exhibits a broad spectrum activity against both gram-positive and
gram-negative bacteria. this antibiotic inhibits folic acid synthesis which prevents
the formation of tetrahydrofolic acid, an essential component for bacterial DNA
synthesis
4. Streptomycin- activity against gram-negative bacteria such as E.coli. This antibiotic
acts by binding to the bacterial 30s ribosomal subunit which interferes with protein
synthesis.
On the other hand, the media used during the selective and differential testing is listed as
follows with their properties.
1. Nutrient agar- supports the growth of a wide range of media commonly used for the
growth of non-fastidious bacteria. It is a nonselective non-enriched and non-
differential general media.
2. Eosin methylene blue agar is a deferential and selective media that is primarily used
for the isolation and differentiation of gram-negative bacteria such as E.coli. It also
differentiates lactose fermenters from non-lactose fermenters. Typically lactose
fermenters form dark purple colonies species that produce high levels of acid will
precipitate the dyes and a metallic green sheen will form. Low-acid fermenters
precipitate a small amount of dye and form pink colonies with dark centres.
3. Mannitol salt agar is a selective and differential medium used for the isolation and
identification of staphylococcus aureus. The high concentration of salt selects for
staphylococcus as it can tolerate high levels of salt. Other organisms is expected to
grow very weakly.
4. Blood agar is an enriched media that contains sheep blood. It supports the growth of
a wide range of microorganisms and provides additional nutrients. Promotes growth
of fastidious organisms. Used to differentiate organisms that lyse blood from those
that don’t. The partial breakdown of red blood cells is observed as clear zones
around colonies and media will appear green and where red blood cells are
completely lysed the medium will appear clear.
Materials and Methodology for antibiotic susceptibility
1. Antibiotic discs containing specific antibiotics
2. Measuring ruler
3. Nutrient agar
4. Inoculation loop
5. Bunsen burner
6. Bacterial Cultures
7. Pipette
Procedure
1. Label nutrient agar with all the relevant cultures used
2. Aseptic collection of 0.1ml of culture broth using a pipette and transferring it to
agar
3. Using a sterilized spreader spread the culture evenly
4. Let dry for 15min
5. Aseptically place antibiotics onto the culture
6. Gently press a sterilized inoculation loop against each antibiotic disc to ensure it
is in contact with culture surface.
7. Incubate for 16-18 hours
8. Measure inhibition zones and interpret results ( S, I, OR R)
Materials and methodology for selective and differential media
1. Inoculation loop
2. Bunsen burner
3. Nutrient agar
4. Eosine methylene blue agar
5. Mannitol salt agar Blood agar
6. Blood agar
Procedure
1. using a marker separate the bottoms of each of the agar plates into 4 sections and
name according to each culture
2. Using a sterile inoculation loop streak a single narrow line of culture to each
section.
3. Incubate and observe results
Results
Table 1: Antibiotic susceptibility results
Diameter
Culture Antibiotic Abbrev (mm) Average CLASS
Escherichia coli Amoxicillin AML10 9 11 11 10 10,5 R
Penicillin P10 11 10 8 10 9,5 R
Sulfamethoxazole RL25 15 18 20 16 17,25 I
Streptomycin S10 18 26 20 21 26,25 S
Staphylococcus aureus Amoxicillin AML10 12 12 15 13 13 R
Penicillin P10 10 9 13 9 10,25 R
Sulfamethoxazole RL25 6 7 10 7 7,5 R
Streptomycin S10 20 21 22 21 21 S
Pseudomonas
aeruginosa Amoxicillin AML10 18 16 14 15 15,75 R
Penicillin P10 14 13 12 12 12,75 R
Sulfamethoxazole RL25 18 26 19 20 20,75 S
Streptomycin S10 26 22 23 24 23,75 S
Bacillus cereus Amoxicillin AML10 14 13 14 14 13,75 R
Penicillin P10 12 16 15 11 13,5 R
Sulfamethoxazole RL25 12 12 8 9 10,25 R
Streptomycin S10 29 23 26 25 25,75 S
Table 1 shows the results for each of the antibiotics results for classification based on the
interpretation of inhibition zones of test cultures. S indicates susceptibility, R shows
resistance and I shows intermediate susceptibility .
Discussion of results of antibiotic susceptibility
Amoxicillin antibiotics are seen to be resistant to all our cultures as indicated by the average
of the inhibition zones this is an indication that microbial pathogens have developed a
resistance towards these antibiotics and is essentially seen to be least effective for
treatment of the above-listed pathogens as per the results. Existing data however indicates
that amoxicillin is a broad-spectrum antibiotic and should be effective against these
pathogens therefore results may be subject to these specific samples and not the overall
population of these bacterial groups.
Penicillin is a narrow-spectrum antibiotic and so therefore we would expect it to be the
least effective against a variety of pathogens. As seen in the results all four cultures are seen
to be resistant to this particular antibiotic which is expected according to literature as there
is seen to be an increase in resistance towards these antibiotics.
Sulfamethoxazole is seen to be intermediately susceptible to E.coli and susceptible to
Pseudomonas aeruginosa. Typically E.coli is seen to be susceptible to this antibiotic
therefore results are supported by literature. However, Pseudomonas aeruginosa which is
known to have resistance mechanisms is susceptible to this antibiotic therefore for this local
group of bacteria therefore serving as an effective antibiotic treatment.
The last antibiotic Streptomycin appears to be the most effective treatment for all of the
cultures used during the experiment indicating susceptibility results each time with large
inhibition zone measurements.
Selective and differential media results
Figure 1 blood agar result description: (1) E.coli - shows a broad growth which
indicates successful growth on medium as it has grown the most of the 3 (2) bacillus
cereus is dark brown and almost makes media see through also has the second
largest growth - (3) S. aureus- shows very minimal growth and pale white in colour
with a more defined boarder. Has a more even surface (4) S. epidermis- white in
colour with an irregular shape around its boarders and varies in surface heights
Figure 2 Eosin methylene blue agar results: (1) S. aureus- has very little growth and
pale brown colour (2) bacillus cereus no growth occurred at all only a streak line
visible (3) S. epidermis appears pink in colour and has an irregular shape along
boarders has an even surface (4) E.coli has a green metallic sheath and appears
brown in colour
Figure 3 nutrient agar: (1) S. aureus appears to have a yellow-white colour and only
growth along the streak line with an irregular boarder (2) Bacillus cereus appears off-
white in colour to have grown just over the streak line with irregular boarders. (3) S.
epidermis appears to be an off-white colour with growth only along a streak line with
an irregular boarder (4) E.coli appears to have a greyish-white colour with growth
only along a streak line
Figure 4 Mannitol salts agar: (1) S. aureus – causes a yellow colour change in the
agar and appears to have a pale yellow appearance with growth occurring only along
the streak line (2) bacillus cereus appears to have a grey-white colour with growth a
little beyond the streak line (3) S. epidermis appears pale white in colour with growth
only along streak line (4) E.coli has a grey-white colour with a waxy or shiny surface
of uneven growth a little beyond streak line
Discussion for selective and differential media
1. Nutrient agar- supports the growth of a wide range of media commonly used for the
growth of non-fastidious bacteria therefore we see growth of all the bacteria on this agar
media with even growth between the 4 which is expected as per the properties of nutrient
agar
2. Eosin methylene blue agar is a metallic green sheen formed around E.coli culture which
indicates high levels of acid during fermentation. Low-acid fermenters precipitate a small
amount of dye and form pink colonies with dark centres as seen with the rest of the cultures
on the media.
3. Mannitol salts agar as seen by the results of a yellow colour change in media is indicative
of staphylococcus aureus which it selects for. The high concentration of salt selects for
staphylococcus as it can tolerate high saline levels of salt. Other organisms are expected to
grow very weakly. When S. aureus grows on this medium it ferments the mannitol salts and
releases acid by-products which change the pH of the phenol red indicator observing a
colour change in order to differentiate between S. aureus and S. epidermis
4. Blood agar is Used to differentiate organisms that lyse blood from those that don’t
therefore we can see that The breakdown of red blood cells is observed as clear zones as a
result of the bacillus culture which indicates cells are completely lysed as the media appears
clear. Where red blood cells are partially lysed the media will appear green as seen with
E.coli and S epidermis.
Conclusion
Finally, antibiotic susceptibility testing is critical for determining optimal antibiotic therapy
and discovering bacterial resistance for local culture samples. By assessing the sensitivity of
bacterial isolates to specific antibiotics, better treatment options can be made and
antibiotic-resistant strains can be avoided. The results outlined for our experiment indicates
the importance of testing bacterial sensitivity for each isolate in order to make the best
treatment options. Selective and differential media, such as Nutrient Agar, Eosine
Methylene Blue Agar, Mannitol Salt Agar, and Blood Agar, can be used to isolate, identify,
and differentiate bacterial species depending on their growth properties and metabolic
activities. The results obtained from experimental procedures are indicative of a successful
and reliable outcome as it is in correlation with existing literature and a range of there
scholarly results obtained by researchers.
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