Production of Antibiotics



by
Ahmed samy

In science faculty in Tanta

university
1- Introduction

2-materials and method

·Materials and methods are shown as the follows

Instruments

Flasks, Petri-dishes ,test tubes ,swabs, bacterial loop and alcohols

Sterilization of instruments

Sterilize the instruments in the oven at 150-180 c for 2-3 hrs and calculate the time after the pointer reach to 150 c

Preparation of nutrient agar medium ( solid media)

·The composition of nutrient agar media is

3gm beef extract

3gm peptone

5 m Nacl

15-20 gm agar

1000 ml dis H2o

pouring the media in Petri-dishes

after sterilization of solid media, pour it on number of Petri-dishes under sterile conditions

isolation of bacteria from different sources and making a cultures

1-isolate of bacteria from different sources that include {air ,soil ,leaf , water ,nail ,hair , finger scratch(skin), coca } and this is achieved as the follows

a) Air sample : by opening the Petri-dish for 10-15 min

b) Soil sample: by taking the soil sample (by digging at the distance of 2 cm) and putting it in Petri –dish

c) leaf sample: by breaking the sample and making a suspension ( 1-2gm in 10ml H20) then putting drop in Petri dish

d) water sample: by taking 0.5 ml of water and drooping it on Petri dish

f) nail sample : by taking part of nail and putting it on Petri-dish

g) hair sample : by taking a hair and putting it on Petri-dish

k) finger scratch sample: by taking scratch from any one finger and putting it on Petri-dish

m) coca sample : by putting drop on Petri-dish

2-incubate the Petri-dishes at incubator at 30-37 c for 72 hrs (2-3 days)

3- after 72hrs observe the contents of Petri-dishes and describe the bacterial colonies according to

a-color : for example yellow ,red etc)

b-size: ( for example 1 mm , 2mm,3mm ,etc)

c-form(shape of colony) : ( for example round(circular) , irregular ,filamentous ,rhizoid , curled)

d-Margin(edge) : (for example entire ,filamentous(filiform) ,undulate , ,lobate ,etc)

f- Elevation (for example raised , flat, convex ,umbonate ,growth into medium ,etc)

g-surface ( for example smooth , rough etc)

h0texture (for example dry, moist ,butyrous , etc)

[IMG]file:///C:/DOCUME%7E1/as/LOCALS%7E1/Temp/msohtml1/01/clip_image004.jpg[/IMG]

(See table 1)

Subculture (purification)

1-Make subculture to each colony in new Petri-dish while every culture is subcultured in bew Petri dish

2-incubate the subculutures at 37c for 2-3 days

3- take each organism and make gram stain

Gram staining

Make the bacterial smear on slide and then make the follows

1-cover the smear with crystal violet for 60 sec

2-wash the slide by drawing water

3-cover the smear with gram’s iodine solution for 60 sec

4-wash the slide by drawing water

5-Decolourize the slide briefly by adding acetone (2-3 seconds).

6 -wash the slide by drawing water

7- add safranin counterstain for 2 min

8- wash the slide by drawing water and olace the slide to dry in air then examine it under microscope

·In Gram-positive bacteria, the dark purple crystal violet stain is retained by the thick layer of peptidoglycan which forms the outer layer of the cell.
In Gram-negative bacteria, the thin peptidoglycan layer in the periplasm does not retain the dark stain, and the pink safranin counterstain stains the peptidoglycan layer.

( See table 2)

Preparation of nutrient broth media (liquid media)

1-Prepare nutrient medium without agar 3gm beef extract

3gm pepton

5gm Nacl

1000ml dis H2o

2-pour the media to fill 1/3 the test tube

3-repeat the last step in number of test tubes

4-sterilize the tubes in autoclave at 121c and 1.5atm for 20 min

5-make numbering to the tubes

6-inoculate the tubes with organisms that we get from pure culture and we want to know if these organism secret compounds that have antimicrobial activity or not

7-get pathogenic(E.coli and bacillus) bacteria and inoculate them in a new plates then make well in the center of the Petri-dish

8-put 0.5 ml from suspension of liquid media that found in tube on the well that found on the center of Petri-dish

9-repeat the last step on different test tubes and different Petri-dish and in every time we put discs of known antibiotics (penicillin ,cephaloexin) to compare between the effects of known antibiotics and unknown antibiotics produced by unknown bacteria

10-incubate the plates in incubator at 37c for 2-3 days

11-get out the Petri-dishes from incubator and motice is there inhibition zones formed around the well or no



12-measure the inhibition if it formed around the wells and measure also the inhibition zones around the two antibiotics(pencillin, cephaloexin) and compare between them ( see table 3)