CONTROL OF MICROBIAL GROWTH AND ANTIMICROBIAL

AGENTS
Antimicrobial agents are drugs, chemicals or other substances that kill or inhibit the
growth oI microorganisms such as bacteria, Iungi or protozoans. Antimicrobial drugs
either kill microbes (microbicidal) or prevent the growth oI microbes (microbistatic).
DisinIectants are antimicrobial substances used on non-living objects. Among the
antimicrobial agents are antibacterial drugs, antiviral agents, antiIungal agents, and
antiparasitic drugs.
A. Antibacterial agents:
acteriostatic antibiotics limit the growth oI bacteria by interIering with bacterial
protein production, DNA replication, or other aspects oI bacterial cellular metabolism.
ut bactericidal agents that totally kill the bacteria. acteriostatic agents must work
with the immune system to remove the microorganisms Irom the body. High
concentrations oI some bacteriostatic agents are also bactericidal, whereas low
concentrations oI some bactericidal agents are bacteriostatic.
1. Tetracycline: %etracycline antibiotics are protein synthesis inhibitor,
inhibiting the binding oI aminiacyl-tRNA to the m-RNA-ribose complex.
%hey do so mainly by binding to the 30S ribosomal sub unit in the m-RNA
translation complex. %etracyclines also have been Iound to inhibit matrix
metalloproteinases. It also binds to the 16S part oI the 30S ribosomal subunit
and prevents the amino-acyl tRNA Irom binding to the A site oI the ribosome.
2. Sulfonamide: It is the basis oI several groups oI drugs. %he original
antibacterial sulIonamides (sometimes called simply sulIa drugs) are synthetic
antimicrobial agents that contain the sulIonamide group. In bacteria,
antibacterial sulIonamides act as competitive inhibitor oI the enzyme
dihydropteroate (DHPS), an enzyme involved in Iolate synthesis.
3. Spectinomycin: It is an aminocyclitol antibiotic, closely related to the
aminoglycosides, produced by the bacterium Streptomyces spectabilis.It binds
to the 30S ribosomal subunit in invading bacteria and interrupts protein
synthesis.It is used to treat gonorrhea, especially in patients who are allergic to
penicillin.
4. Trimethoprim: %his antibiotic mainly used in the prophylaxis and treatment
oI urinary tract inIections. It belongs to the class oI chemotherapeutic agents
known as dihydroIolate reductase inhibitors which inhibit synthesis oI
tetrahydroIolic acid.
5. Chloramphenicol: It is a bactericidal antimicrobial agent. Chloramphenicol is
eIIective against a wide variety oI Gram-positive and Gram-negative bacteria.
%he original indication oI chloramphenicol was in the treatment oI typhoid
and also used as a second-line agent in the treatment oI tetracycline-resistant
cholera. Chloramphenicol is active against the three main bacterial causes oI
meningitis: eisseria meningitides, Streptococcus pneumoniae and
Haemophilus influen:ae. %he most serious adverse eIIect associated with
chloramphenicol treatment is bone marrow toxicity.
6. Macrolides:%hese are a group oI drugs (typically antibiotics) whose activity
stems Irom the presence oI a macrolide ring, a large macro cyclic lactone ring
to which one or more deoxy sugars, may be attached. etolides are a new
class oI antibiotics that are structurally related to the macrolides. %hey are
used to Iight respiratory tract inIections caused by macrolide-resistant
bacteria. Ketolides are especially resistant as they have a double-binding
site.Macrolides are protein synthesis inhibitor and they are thought to do this
by preventing peptidyl-transIerase Irom adding the peptidyl attached to tRNA
to the next amino acid as well as inhibiting ribosomal translocation.Antibiotic
macrolides are used to treat inIections caused by Gram positive bacteria,
Streptococcus pneumoniae, and Haemophilus influen:ae inIections such as
respiratory tract and soIt tissue inIections.
7. Lincosamides: It is (eg. Lincomycin and clindamycin) are a class oI
antibiotic.Lincosamides are protein synthesis inhibitors which bind to the 23s
portion oI the 50S subunit oI bacterial ribosomes and cause premature
dissociation oI the peptidyl-tRNA Irom the ribosome. %hey are normally used
to treat StaphylococciandStreptococci. %hey are used in the treatment oI Toxic
Shock Syndrome and thought to directly block the M protein production that
leads to the severe inIlammatory response.
. Antiviral agents: Most oI the antivirals now available are designed to help deal
with HIV, herpes viruses, best known Ior causing cold sores and genital herpes.
Antiviral drugs work by inhibiting the virus beIore it enters the cell, stopping it
Irom reproducing or in some cases, preventing it Irom exiting the cell.
C. Antifungal agents: An antiIungal drug is used to treat Iungal inIections such as
ringworm, candiasis, serious systemic inIections such as cryptococcal meningitis,
and others. AntiIungals work by exploiting diIIerences between mammalian and
Iungal cells to kill oII the Iungal organism without dangerous eIIects on the host.
D. Antiparasitic antigens: Antiparasitics are a class oI medications which are
indicated Ior the treatment oI inIection by parasites such as nematodes, cestodes,
trematodes, inIectious protozoa and amoebae. Like antiIungals, they must kill the
inIecting pest without serious damage to the host.


!RACTICAL-19
Aim: Determination of minimum inhibitory concentration (MIC) of different
antimicrobial agents.
Materials required: Antimicrobial agents, L broth medium (tryptone- 10 g/L,
yeast extract- 5 g/L, NaCl- 5 g/L and distilled water- 1 L), culture tubes,
spectrophotometer, test tubes, bacterial culture, inoculating loop and incubator.
!rinciple: %he smallest amount oI antibiotic necessary to inhibit a test organism
is known as minimum inhibitory concentration oI antibiotic (MIC) which may
inhibit the growth oI a particular bacterium. y supplementation oI streptomycin
antibiotic, the protein synthesis oI bacteria hampers because streptomycin binds
30S subunit oI ribosome by which ribosome becomes inactive. %hereIore cells
stop dividing due to check oI new protein synthesis and lose viability.
!rocedure:
1. Prepare the required concentrations oI antimicrobial/antibiotic solutions by
making serial dilutions Irom the stock one.
2. %ake seven bacterial culture tubes in which pour 5 ml oI L media in each.
3. Inoculate all the tubes with 20-30 l oI prepared bacterial culture.
4. Add diIIerent concentrations oI antibiotic/antimicrobial solution in all the
tubes except control one (L broth media bacterial culture).
5. Now incubate all the tubes at 30
0
C Ior 24 hours.
6. %hen measure turbidity in terms oI optical density(O.D.)at 610 nm and plot a
graph taking concentrations oI antibiotic/antimicrobial solution on X-axis
against absorbance at 610 nm on Y-axis.
Observations:
1. Observe the growth oI bacteria by means oI appearance oI turbidity in each
tube.
2. Note down O.D. oI the bacterial culture oI each tube correctly. Low conc. OI
antibiotic will show maximum O.D. whereas high conc. will show minimum
O.D.
Result and discussion:
!recautions:



!RACTICAL-20
Aim: Test for antibiotic sensitivity of Gram positive /Gram negative bacteria
by Disc method (irby-Bauer method).
Materials required: L agar medium (tryptone- 10 g/L, yeast extract- 5 g/L,
NaCl- 5 g/L, agar- 18 g/L and distilled water- 1 L), bacterial culture (Gram ve-
acillus thuringiensis and Gram ve- Salmonella typhimurium, E.coli), diIIerent
antibiotic discs, petri plates, spreader, unsen burner and incubator.
!rinciple: ased on whether a particular bacteria are susceptible to speciIic
antibiotics or not and the eIIectiveness is based on size oI inhibition zone. More
the zone oI inhibition more is the sensitivity less is the zone less eIIective is the
antibiotic. II no zone is seen the bacteria is resistant to antibiotic.
%he drugs used in the medical sciences include antibiotics, sulphonamides, and
chemotherapeutics which are antimicrobial in nature.Antibiotic sensitivity is quite
signiIicant due to development oI resistance among various microorganisms. %he
sensitivity oI the drug helps in selecting the appropriate line oI treatment.
!rocedure:
1. Prepare agar medium and autoclave it.
2. %ake two petri plates, one Ior Gram positive bacteria and another Ior Gram
negative bacteria and pour the agar medium in to petri plates and leave to
solidiIy.
3. %hen spread one plate with overnight cultured Gram ve bacteria and another
with Gram -ve bacteria.
4. %hen place diIIerent antibiotics discs on both plates and incubate the plates at
37
0
C Ior overnight.
Observations:
1. Examine the overnight incubated plates and measure the diameter oI the
clearing zones in centimetre.
2. %hen make a table by taking name oI antibiotics in one column and zone oI
sensitivity (in cm.) in another column.
3. %he Iaint growth or tiny colonies in the clearing zone may appear due to
resistant nature some bacteria. Avoid such growth.
Result and discussion: