Topic: antimicrobial and chemotherapy

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The sources must be updated at least from 2005-2014 . From credible sources books and journals. For each answers I need at least 1 reference with the Page No. And in

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Answer the questions separately (not in essay style).
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( You must include 5 sources including the recommended 3 sources below):-

1- Antibiotic and Chemotherapy: Anti-infective Agents and Their Use in Therapy, Eighth Edition

2- Residual moisture determines the level of touch-contact-associated bacterial transfer following hand washing. Patrick DR1, Findon G, Miller TE.

3- Kucers
The use of antibiotics sixth edition

SESSION 6: PROPERTIES OF ANTIMICROBIAL SUBSTANCES
MODE OF ACTION OF BENZYLPENICILLIN ON E. coll W.
INTRODUCTION
The synthesis of the rigid cell wall components in both gram-positive and gram-
negative bacteria can be effected by the presence of antibiotics. The beta-lactam
groups of antibiotics (e.g. penicillins and cephalosporins) interfere with wall synthesis
by actions on the various transpeptidases found in bacteria. The interference can be
monitored morphologically using phase-contrast or bright-field microscopy and the
concentration dependent effects will be demonstrated in this practical. The absorbance
will be monitored to determine the time taken for the full lysis to become evident.

A. SETTING-UP AND SAMPLING

Thls part of the experimental work will he carrlecl out by the Technical Staff and the

samples provlcled for the lnvestlgallons.

1. inoculate about 6 mL of an overnight culture of E. collW into 10-12 mL of TSB in
a sterile 50 mL conical flask and incubate in the shaking water bath at 37° for
one hour. This produces the bacterial suspension referred to in the table below.
This suspension will be provided by the staff.

2. Prepare the following experimental systems (A, B, C, D, E and F) in 50 mL
sterile conical flasks. All the solutions are provided sterile and are to be handled
with ASEPTIC technique. The quantities are in mL.

A B C D E F

TSB* 12 0 12 0 12 0

TSB and stabiliser** 0 12 0 12 0 12

Benzylpenicillin (10,000 UlmL) 0 0 2 2 0 0

Benzylpenicillin ( 1,000 UlmL) – – – – 0.5 0.5

Millipore Pure Water (MPWJ 2 2 0 0 1.5 1.5

E. collsuspension (to be added last) 6 6 6 6 6 6

TSB is Trypticase Soy Broth or SCDM Soy Casein Digest Medium.

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Stabiliser consists of 0.45M sucrose plus 0.011l‘v1 magnesium sulphate (or as

specified).

3. Immediately on completion of setting up the flasks by adding the bacterial
suspension take a zero time sample by removing 2 mL of the culture system and
transferring to a Bijou bottle containing 0.1 mL formalin. Rinse the pipette in
boiling distilled water and rinse in saline between sampling of the various
systems. Keep the same pipette and saline for each sampling.
4. Incubate the flasks fer 100 minutee, with cnly gentle ahal-rlng, at 37°C. At each
successive 25 minutes remeve 2 mL frcm each culture evetem and transfer tc
eeparate bettlee ccntaining 0.1 mL fermalin. Examine macreeccpically after the
laet eampling time and reccrd.

B. INVESTIGATIONS {Seeeicn 6]

1. Prepare an agar-film meunt frem each eample tc be inveetigated and examine
theee ueing a phaee-cc-ntraet micreecepe and the eil-immereicn et:-jective.
Initially examine the ccntrel eeriee B at ‘(’5 minutes and than the teat eeriee D
and F at 50 minutes. Once veu have identified the aberrant fc-rme, mcve
ferward and back in the time eeriee tc identify earlier and later effecte in the
eveteme. If time permits examine the later time eamplee ef eeriee E. [The data
will ehared within the class]

Twe phaee-ccnlrael rnlcreecc-pee wlll be available.

2. The abeerbance ie determined using the Ivlatertek SF’-830 Spectrephetemeter at
550 nm. Dilute the eamplee te be checked with TSB + etabilieer (1.5mL + 1.5mL)
and uee TSB + etabilieer (9 mL + 1.0 mL water} ae a blank. Te eccnemiee en
dilutien ccntainere dc ene experimental evetem at a time, then rinee and drain
the centainere befere re-uee.

(mine)
22

RESULTS

1. Sketch the appearance ef repreeentative eamplee as viewed, ueing the
micrcecepe. Preeent a eeguential erdering ef the merphelegical changee
evident, ueing the ccllated material frem the whitebcard. Label the figuree.

2. On a eingle graph, plet the aheerbance ef each evetem against time.

AGAR-FlLl‘v’l MOUNT

{a) Drcp abcut 0.5 mL ef melten agar {1-1.5%) en the eurface cf a level micreecepe
elide and allcw te eet.

{I3} With the edge cf a elide trim the agar film te an apprex. 1 cm equare in the
middle ef the elide.

{cl Place a lecpful cf the euepeneicn tc be examined in the middle cf the agar
equare. Carefully’ cever with a ceverelip.

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