زيزو1900
03-04-2010, 10:11 PM
Production of Antibiotics
by
Ahmed samy
In science faculty in Tanta
university
1- Introduction (http://www.comp.leeds.ac.uk/roger/HiddenMarkovModels/html_dev/main.html)
Antibiotics are chemicals produced or derived from microorganisms (i.e. bugs or
bacteria (http://en.wikipedia.org/wiki/Bacteria)
germs such as bacteria and fungi) to kill or inhibit the growth of
antibiotics also belong to the broader group of antimicrobial (http://en.wikipedia.org/wiki/Antimicrobial) compounds, used to treat infections caused by microorganisms (http://en.wikipedia.org/wiki/Microorganism), including fungi (http://en.wikipedia.org/wiki/Fungus) and protozoa (http://en.wikipedia.org/wiki/Protozoa)
Some antibiotics are 'bactericidal', meaning that they kill the growth of bacteria. While Other antibiotics are 'bacteriostatic', meaning that they inhibit the growth of bacteria
different type of antibiotic affects on different bacteria in different ways. . For example, an antibiotic might inhibit ability of bacteria to turn glucose into energy, or its ability to construct its cell wall. When this happens, the bacterium
dies instead of reproducing.
Some antibiotics can be used to treat a wide range of infections these known as 'broad-spectrum' antibiotics while Others are only effective against a few types of bacteria and are called 'narrow-spectrum' antibiotics (target gram-negative bacteria only or gram-positive bacteria)
The effectiveness of individual antibiotics varies with the location of the infection and the ability of the antibiotic to reach this site.
Oral antibiotics are the simplest approach when effective but intravenous antibiotics reserved for more serious cases while Antibiotics may sometimes be administered topically, as with eye drops or ointments.
the first antibiotic was penicillin that discovered by Alexander Fleming in 1928 in a significant breakthrough for medical science
The main classes of antibiotics: file:///C:/DOCUME%7E1/as/LOCALS%7E1/Temp/msohtml1/01/clip_image002.jpg
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
after preparation of media , sterilize the media( solid media ) in the autoclave at 121 c and 1.5 atm for 20 min
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)
file:///C:/DOCUME%7E1/as/LOCALS%7E1/Temp/msohtml1/01/clip_image004.jpg
(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)
by
Ahmed samy
In science faculty in Tanta
university
1- Introduction (http://www.comp.leeds.ac.uk/roger/HiddenMarkovModels/html_dev/main.html)
Antibiotics are chemicals produced or derived from microorganisms (i.e. bugs or
bacteria (http://en.wikipedia.org/wiki/Bacteria)
germs such as bacteria and fungi) to kill or inhibit the growth of
antibiotics also belong to the broader group of antimicrobial (http://en.wikipedia.org/wiki/Antimicrobial) compounds, used to treat infections caused by microorganisms (http://en.wikipedia.org/wiki/Microorganism), including fungi (http://en.wikipedia.org/wiki/Fungus) and protozoa (http://en.wikipedia.org/wiki/Protozoa)
Some antibiotics are 'bactericidal', meaning that they kill the growth of bacteria. While Other antibiotics are 'bacteriostatic', meaning that they inhibit the growth of bacteria
different type of antibiotic affects on different bacteria in different ways. . For example, an antibiotic might inhibit ability of bacteria to turn glucose into energy, or its ability to construct its cell wall. When this happens, the bacterium
dies instead of reproducing.
Some antibiotics can be used to treat a wide range of infections these known as 'broad-spectrum' antibiotics while Others are only effective against a few types of bacteria and are called 'narrow-spectrum' antibiotics (target gram-negative bacteria only or gram-positive bacteria)
The effectiveness of individual antibiotics varies with the location of the infection and the ability of the antibiotic to reach this site.
Oral antibiotics are the simplest approach when effective but intravenous antibiotics reserved for more serious cases while Antibiotics may sometimes be administered topically, as with eye drops or ointments.
the first antibiotic was penicillin that discovered by Alexander Fleming in 1928 in a significant breakthrough for medical science
The main classes of antibiotics: file:///C:/DOCUME%7E1/as/LOCALS%7E1/Temp/msohtml1/01/clip_image002.jpg
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
after preparation of media , sterilize the media( solid media ) in the autoclave at 121 c and 1.5 atm for 20 min
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)
file:///C:/DOCUME%7E1/as/LOCALS%7E1/Temp/msohtml1/01/clip_image004.jpg
(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)