TRIPLE SUGAR IRON

Procedure

1- with the help of a sterilized wire loop pick up a well isolated colony from medium.

2- stab the wire in the center of TSI medium .

3- then inoculate it bt making a zigzag position with wire loop on the slant of agar.

4- incubate the tube at 37C for 24 hours.

5- observe the results carefully. Accessed:19-12-16

Results:

If the lactose ferment by the organism present then butt and slant of medium turned yellow.

If the lactose not fermented then butt is turned into yellow while slant becomes red.

If the glucose lactose and sucrose not fermented both butt and slant turned red. Accessed:19-12-16

See the figures

Shows positive TSI test

Shows positive TSI test 

CITRATE TEST

Procedure

1-Pour the bacterium into simmons citrate medium.

2-Incubate at 37^oC . for 24 hours.

3-Observe the results. Accessed:16-12-16

Results

No change in medium indicates negative. Figure5

Blue color production indicates test result positive.Figure6 Accessed:16-12-16

Show Negative Citrate test

Show Positive Citrate test

UREASE TEST

Principle:

Many organisms especially which infect the urinary tract are able to split urea into carbon dioxide and ammonia in the presence of water. Ammonia combines with both water and carbon dioxide and produces the ammonium carbonate. This ammonium carbonate changes the pH of medium to alkaline and produces a pink color from original yellow color.Accessed:16-12-16

Procedure

1-Pour the pure bacteria suspension into the broth medium.

2-Loosely tight the cap of tube and leave at 35 °C ambient air.

3-Observe the result. Accessed:16-12-16

Result

No change considered as negative result. Figure 3

Color change into pink will be indicated the result as positive test. Figure 4

Accessed:16-12-16

Shows Negative Urease test

Shows Positive urease test

BIOCHEMICAL TEST

Indol

Indol test is a biochemical test which are performed for the detection of bacterial species which are having the ability of converting the amino acid tryptophan and producing indol. Accessed:12-12-16

Principle of test:

Some bacteria have an enzyme tryptophanase .This enzyme have the ability to split amino acid tryptophan and pyruvic acid and produce the indole. The presence or production of indole can be detected by the Kovac’s reagent or by the Ehrlich’s reagent which gives red colour ring on the top of media tube. Accessed:12-12-16

Procedure

1- Make the suspension of bacterium to be tested in distilled water and pour into typtone broth.

2- Incubate for 24 hours at 37°C.

3- After incubation add few drops of Kovac’s reagent into tube containg broth and bacterium.

Ehrlich reagent is more sensitive for non, fermentors and anaerobes.

4- Observe the result carefully and avoid to shaking the tube. Accessed:12-12-16

Result:

Red or pink color on the top indicated as the Positive test. Figure 2

No change will be resulted as Negative result. Figure 1

Accessed:12-12-16

Shows Negative Indol Test

Shows Positive Indol Test

PUS CULTURE

Initial examination:

The appearance of a specimen of pus and that of any appreciable amount of pus on a swab was observed (Koneman et. al., 2005).

Characterization of Bacterial Isolates:

1.      The pus specimen was inoculated on blood and MacConkey agar plates.

2.      The streaked plates were incubated at 37°C for 24 hr.

3.      Bacterial colonies on blood agar plates were later Gram stained.

4.      Characterization of bacterial isolates was based on standard microbiological methods.

5.      Identification of isolates were done based on colony morphology, motility, catalase test, oxidase test, coagulase test and biochemical tests like Tripal sugar iron agar, Hydrogen sulfide test, Carbohydrate fermentation test, Phenylalanine deaminase test, Methyl red test, Nitrate reduction test, Urease test, Voges proskauer, Citrate utilization test, Indole test (Koneman et. al., 2005).

URINE CULTURE

The quantification of bacteria in urine can be done by several methods. Among them sterile filter paper strip method, calibrated loop technique and multipoint technology are the easiest and routinely used methods (Fallon et. al., 2003) (Haugen et. al., 1968). Of these, multipoint methodology using CLED or chromogenic media, are considered to be the most versatile and efficient for large numbers of specimens. Other methods include use of dipslides, pour plates and roll tubes. (Aspevall et. al., 2002)

Filter paper method

1.      Commercially prepared sterile filter paper strip was dipped in the urine up to the mark indicated. .

2.      Excess or the urine was removing by touching the edge of the strip against the side of the specimen container.

3.      The remaining urine was allowed to absorb into the strip before inoculating a CLED or chromogenic agar plate.

4.      Inoculated end of the strip was Bend and press flat against the agar for a few seconds.

5.      Several specimens were inoculated onto one CLED agar plate (Fallon et. al., 2003).

Figures1,2

Antimicrobial Senstivity:

The Kirby-Bauer (K-B) test utilizes small filter disks impregnated with a known concentration of antibiotic. The disks are placed on a Mueller-Hinton agar plate that is inoculated with the test microorganism. Upon incubation, antibiotic diffuses from the disk into the surrounding agar. If susceptible to the antibiotic, the test organism will be unable to grow in the area immediately surrounding the disk, displaying a zone of inhibition. The size of this zone is dependent on a number of factors, including the sensitivity of the microbe to the antibiotic, the rate of diffusion of the antibiotic through the agar, and the depth of the agar. Microorganisms that are resistant to an antibiotic will not show a zone of inhibition, (accessed 5-12-17)

Shows the Filter paper Urine culture strip

Shows the Bottle of filter paper urine culture strip

PUS SAMPLES

Same as urine samples 50 pus samples from different sites have been cultured from which 24 samples have no growth and 26 samples have growth of different bacteria’s. From 26 samples only 4 samples have ESBL producing bacteria while rest of 22 samples are non ESBL bacteria’s.See figures

Total num of samples	No growth	ESBL	Others
50	24	04	22

Shows result of pus culture

Total ESBL producing bacteria	E.coli	Klebsiella	Others
04	02	00	02

           shows pus culture results of E.coli ESBL and Other ESBL

Show results of Pus culture samples

pus culture results of E.coli ESBL and Other ESBL

URINE SAMPLES

I examined 100 urine samples randomly . after examining I looks that 78 samples have no growth out of 100. 22 samples have growth from which 9 samples have ESBL producing bacteria growth while the other 1 samples have non ESBL producing bacteria. See figures

Total num of samples	No growth	ESBL	Others
100	78	09	13

 Shows the Urine Samples result

Total ESBL producing bacteria	E.coli	Klebsiella	others
09	05	02	02

Urine culture results of E.coli ESBL and Other ES

Shows the Results of urine samples

Urine culture results of E.coli ESBL and Other ESBL

BL

PSEUDOMONAS AERUGINOSA

·         Pseudomonas aeruginosa is gram negative bacteria . its is also aerobic bacteria.

·         Pseudomonas mostly present in any moist location.

·         It is a common cause of nosochomial infections. Nosochomial ifections are those infections which are acquired through hospitals or other family care facility. They are spread thorugh various means.

·         Pseudomonas aeruginosa is also an oppertunistic pathogen . oppertunistic pathogens infects the person when oppertunity is available like patient have weakend  immune system . they are not harmfull normally.(Monica2000)

1.6.1:Pseudomonas aeruginosa infections:

·         They mostly concern with cystic fibrosis , burn patients and hospitilized patients.

·         Mortaility rate of case is approches to 50%.

·         Cystic fibrosis is most common life threating disease. it is caused by the inherited genetic disorder.

·         One in every 25 carries the mutated recessive gene.

·         More than 1 in 4000 live births suffers from cystic fibrosis.

·         Cystic fibrosis affects number of body organs.  Cycle of infection leads to progressive deterioration of lung function.(Monica2000)

1.6.2:Patho mechanism:

·         Pathogenesis starts from adhesion by means of flagella, pili, fimbriae.

·         Extracellular enzymes and toxins help for its invasion.

·         Leuckocidin help it to prevent itself from leukocytes and neutrophills.

·         LPS exotoxin also prevent from immune system.

·         Protection is also carried out by capsule.

1.6.3: Treatment:

·         P.aeruginosa resistant to many commonly used antibiotics but combination of gentamicin and carbeniciilin are frequently used for its treatment.

·         There is vaccine discovered for pseudomonas so far.

(Monica2000)

SHIGELLA

1.      It is gram negative.

2.      Non-motile

3.      Enteric pathogen causing bacillary dysentery

4.      It has four species

a)      Shigella dysenteriae

b)      Shigella flexneri

c)      Shigella boydii

d)      Shigella sonnei

Found in only humans intestinal tract.

It is acquired by ingesting contaminated food and water with shigellae.

1.5.1: Culture characteristics:

1.      Facultative anaerobes

2.      Catalase positive except Shigella dysenteriae

3.      Does not produce H2S

4.      On Blood Agar

5.      2-4mm in size

6.      Entire and convex

7.      On MacConkey Agar

8.      Pale colonies

9.      Non lactose fermenting

10.  Following other media are used

11.  Deoxycholate citrate agar (DCA)

12.  Salmonella Shigellae agar (SS agar)

13.  Xylose lysine Deoxycholate agar (XLD agar).(Monica2000)

Antisera are used for the identification by agglutination reaction.

1.5.2: Pathogenicity

Pathogenesis includes

1.      Bacillary dysentery

2.      Meningism and other neurological symptoms.

3.      Neurotoxins produce by shigellae type-1 which affects central nervous system.

1.5.3: Antibiotic sensitivity

Shigellae susceptible to

1.      Ampicillin

2.      Chloramphenicol

3.      Cotrimoxazole

4.      Tetracycline

5.      Nalidixic acid

6.      Fluoroquinolones (Ciprofloxacin and Ofloxacin).(Monica2000)