Download ANTIBIOTIC

ANTIBIOTIC LAB
Objectives:
•To utilize specific monitoring techniques to evaluate the susceptibility of a microbe to
different antibiotics.
•To identify zones of inhibition for certain bacteria.
•To distinguish the range of activity of an antibiotic.
NOTE ANTIBIOTIC ALLERGIES IN STUDENTS: Please let me know BEFORE we begin this
lab if you have any antibiotic allergies.
As you complete the Antibiotic lab, take notes in your lab notebook.
Background: Most microbiologists distinguish two groups of antimicrobial agents used in the
treatment of infectious disease: antibiotics, which are natural substances produced by certain groups
of microorganisms, and chemotherapeutic agents, which are chemically synthesized. A hybrid
substance is a semisynthetic antibiotic, wherein a molecular version produced by the microbe is
subsequently modified by the chemist to achieve desired properties. Furthermore, some
antimicrobial compounds, originally discovered as products of microorganisms, can be synthesized
entirely by chemical means. In the medical and pharmaceutical worlds, all these antimicrobial agents
used in the treatment of disease are referred to as antibiotics, interpreting the word literally.
The modern era of antimicrobial chemotherapy began in 1929, with Fleming's discovery of the
powerful bactericidal substance, penicillin, and Domagk's discovery in 1935 of synthetic chemicals
(sulfonamides) with broad antimicrobial activity.
In the early 1940's, spurred partially by the need for antibacterial agents in WW II, penicillin was
isolated and purified and injected into experimental animals, where it was found not only to cure
infections but also to possess incredibly low toxicity for the animals. This fact ushered into being the
age of antibiotic chemotherapy, and an intense search for similar antimicrobial agents of low toxicity
to animals that might prove useful in the treatment of infectious disease. The rapid isolation of
streptomycin, chloramphenicol and tetracycline soon followed, and by the 1950's, these and several
other antibiotics were in clinical usage.
Most of the natural antibiotics that are being used in agriculture and medicine are produced by three
unrelated groups of microbes, including eukaryotic molds and two types of spore-forming bacteria.
However, many culturable, and some non culturable microbes, have been shown to produce various
substances that inhibit other organisms that grow in their space. If we consider antibiotics as
secondary metabolites of microbes, it narrows the field to the handful of microbes discussed below.
Penicillium and Cephalosporium molds produce beta-lactam antibiotics such as penicillin and
cephalosporin and their relatives. They also produce the base molecule for development of
semisynthetic beta-lactam antibiotics, such as amoxicillin and ampicillin. Beta-lactams are used to
treat about one-third of outpatients with bacterial infections.
The natural habitat of molds is soil. And although sex is sometimes involved, they reproduce by
spore formation. They are foremost in their abilities to degrade organic matter, and they play their
most important role in natures in biodegradation and the carbon cycle. Most of us know that molds
will grow on nearly anything that is organic and moist, so they are also responsible for a lot food
spoilage as well as decomposition of our structural materials and textiles. "Nothing is forever", with
molds around.
Actinomycetes, mainly Streptomyces species, produce tetracyclines, aminoglycosides
(streptomycin and its relatives), macrolides (erythromycin and its relatives), chloramphenicol,
ivermectin, rifamycins, and most other clinically-useful antibiotics that are not beta-lactams.
Actinomycetes are the mainstay of the antibiotics industry.
Actinomycetes are a group of branched bacteria that reproduce by spore formation. They come from
a phylum of Bacteria, Actinobacteria, and they are landed in Order Actinomycetales. Some of the
representative family include such diverse bacteria as Actinomyces, Corynebacterium, Nocardia,
Propionibacter, Streptomyces, Micromonospora and Frankia. Most actinomycetes are inhabitants of
the soil. The characteristic odor of damp soil is due to the production of substances, called geosmins,
by these bacteria
Bacillus species, such as B. polymyxa and B. subtilis, produce polypeptide antibiotics (e.g.
polymyxin and bacitracin), and B. cereus produces zwittermicin. Bacillus species have the relatively
rare ability to form a type of resting cell called an endospore. Bacilli are Gram-positive, rod-shaped,
aerobic bacteria that live in the soil. They play an important ecological role in aerobic
decomposition, biodegradation and mineral recycling.
These organisms all have in common that they live in soil and they form some sort of a spore or
resting structure. It is not known why these microorganisms produce antibiotics, but the answer may
be in the obvious - it affords them some nutritional or spatial advantage in their habitat by
antagonizing the competition; or it may be in the subtle - it acts as some sort of hormone or signal
molecule associated with sporulation or dormancy or germination. Antibiotics are secondary
metabolites and they are produced at the same time that the cells begin their sporulation processes.
Antibiotics tend to be rather large, complicated organic molecules and may require as many as 30
separate enzymatic steps to synthesize. The maintenance of a substantial component of the bacterial
genome devoted solely to the synthesis of an antibiotic leads one to conclude that the antibiotic is
important, if not essential, to the survival of these organisms in their natural habitat.
Most of the microorganisms that produce antibiotics are resistant to the action of their own
antibiotic, although the organisms are affected by other antibiotics, and their antibiotic may be
effective against closely-related strains. In most cases, how or why bacteria are resistant to their own
antibiotics is also unknown, but it may be worth pondering or studying if we are to understand the
cellular and molecular basis of drug resistance in pathogens.
Todar, PhD, K. (2008). Antimicrobial Agents in the Treatment of Infectious Disease. Retrieved June
30, 2015.
Part I: Click on the link http://www.microbiologyonline.org.uk/about-microbiology/microbes-andthe-human-body/antibiotics and read the information listed under Antibiotics.
Part II:
Evaluating the Effectiveness of Antimicrobial Agents:
The Disk-Diffusion Method
The disk diffusion method is used to evaluate the antimicrobial efficacy of chemical agents. A
sterile disk of filter paper or cardboard is soaked with a chemical and placed on a bacterial lawn.
After incubation, if the chemical is effective, a clear zone called a zone of inhibition can be seen
around the disk.
Objective:
The student will evaluate the antimicrobial effectiveness of various chemicals
against a Gram positive and a Gram negative species.
Materials for 3 or 4 students
Cultures
2 Petri dishes with nutrient agar
12 sterile disks
forceps
bacterial spreader
culture dish with ethanol
Bunsen burner
Micropipette and sterile tips
Bacillus cereus (Gr. +)
Escherichia coli (Gr. -)
1) Creating two bacterial lawns
At the center lab table
It will take 2 students to create each culture. First, you will need to carefully place a sterile
tip on the micropipette. ASEPTICALLY, one student will remove the stopper from the first
culture. While the second students holds the Petri dish lid over the agar at an angle, the first student
will carefully transfer a 1mL sample of broth culture onto the agar. The dish is then closed. The
pipettes are disposed of in bleach water.
This is repeated with the second plate and the other culture.
Spreading the culture at your lab table
Again, two students are needed. You will sterilize the bacterial spreader by placing the triangular
end in the ethanol. Carefully, this is then lit using the Bunsen burner.
**DO NOT LEAVE THE SPREADER IN THE FLAME.
IT WILL BURN YOU!
**KEEP THE SPREADER TRIANGLE BELOW YOUR
HAND SO THE BURNING ALCOHOL DOES NOT DRIP ONTO YOUR HAND!
**DO NOT IGNITE THE BOWL OF ETHANOL!!!!
While one student holds the Petri dish lid at an angle, the other student will use the spreader to
evenly distribute the culture over the agar surface. Be careful not to tear the agar by pressing down
too hard.
The spreader is again sterilized, and this is repeated for the second culture.
2) Applying the Antimicrobial Disks
MARK THE BOTTOM OF THE DISH AS SHOWN BELOW IN ORDER TO KNOW WHICH
CHEMICAL IS ON EACH DISK. Your group will choose 5 chemical agents to study. The same 5
agents will be used on each dish. Each disk is prepared by flaming the forceps. These are used to
grab a sterile disk, and then it’s dipped into the desired chemical. The disk is then placed on the agar
and gently pressed into place. When all 5 disks are in place, the plates are incubated inverted for
24-48 hours.
1=
2=
3=
4=
5=
3) Incubate plates for 48 hours.
Part III: Based on the table below, which antibiotics were effective against your organism?
ANTIBIOTIC
Erythromycin
Tetracycline
Penicillin
Neomycin
Streptomycin
Novobiocin
Kanamycin
Chloramphenicol
CODE
E
Te
P
N
S
NB
K
C
Diameter of Zone of Inhibition in mm
Bacillus cereus
E. coli
Antibiotic
Abbreviation
on disc
Resistant
Diameter
Intermed
Diameter
Sensitive
Diameter
Streptomycin
S
11 mm
12-14 mm
15 mm
Penicillin
P
14 mm
14-16 mm
17 mm
Erythromycin
E
13 mm
14-22 mm
23 mm
Chloramphenicol
C
12 mm
13-17 mm
18 mm
Tetracycline
Te
14 mm
15-18 mm
19 mm
Neomycin
N
12 mm
13-16 mm
17 mm
Novobiocin
NB
11 mm
12-15 mm
16 mm
Kanamycin
K
13 mm
14-17 mm
18 mm
1) Which antibiotics were effective against BOTH species?
2) Did any of the antibiotics affect the two species differently?_____________
If so, explain why this might occur._____________________________________
____________________________________________________________
____________________________________________________________
3) Use your textbook or the internet to research HOW (the mechanism) each antibiotic works.
Explain each in lab notebook.