University of Kerbala/ College of Medicine Department of Microbiology

Unit 2/ Lab 1

1^st year, 2024-2025

م.م. رنا طالب جواد

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Gastrointestinal tract infection (Gastroenteritis)�

�Gastrointestinal tract infection (Gastroenteritis)�

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• Gastroenteritis: Gastroenteritis, also known as infectious diarrhea and gastro, is inflammation of the gastrointestinal tract including the stomach and intestine.
• Symptoms may include diarrhea, vomiting, and abdominal pain. Fever, lack of energy, and dehydration may also occur.

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Gastrointestinal tract infection (Gastroenteritis)

• Diagnosis:
• General & microscopic Stool Examination
• 1. Stool examination for the presence of WBCs and RBCs.
• 2. Fresh stool for ova and parasite.
• 3. There is Giardia lamblia’s rapid antigen detection test.
• 4. E. Histolytica can be seen by microscopy or antigen detection.
• 5. The serum antibody test for an amoebic liver abscess.

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�Diagnosis �

Diagram (1): Diagnosis of Diarrhea

Figure3:Giardia lamblia Figure4: Entamoeba histolytica cyst

Figure 2: Rotavirus Antigen in Human stool

Figure 1:  Gram stain of C. jejuni from pure bacterial culture

Enterobacteriaceae

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Laboratory diagnosis

• Eosin methylene blue (EMB), MacConkey agar
• IMViC test
• TSI test
• Motility test
• Urease test

Test for lactose fermentation:

Eosin Methylen blue EMB agar:

• Eosin for inhibition of G+ve organism growth.
• Only sugar is lactose; if the organism have enzyme that allow lactose fermentation, then acid is produced that leads to change in PH and result in colored colonies. Alternativally, Peptone is the energy source, if the organism is unable to ferment lactose and result in colorless colonies.

MacConkey agar:

• High bile content inhibits the growth of G+ve organism
• If lactose fermented, change in PH and result in colored colonies, If not - peptone is the energy source for growth of organism and result in colorless colonies.

macconkey agar

E.coli on Eosin methylene blue (EMB)

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IMVIC TEST

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Indole test

• a tryptophan
• The bacteria that have the ability to breakdown tryptophan for nutritional need using enzyme called tryptophanase.
• When tryptophan is brokendown, the presence of indole can be detected by the use of Kovacs reagent, which is yellow, react with endole, red color on the surface of the tube.
• Shigella spp Positive for this test, Salmonella
• Ability of bacteria to utilize citrate as its only source of carbon (simmons citrate media)

Indole test�

�Methyl red- Voges Proskauer test�

Methyl red- Voges Proskauer test

• MR-VP broth tube:
• Glucose fermentation pathway:●Enterobacteriaceae can breakdown glucose to pyruvate. The two major pathways for further breakdown of pyruvate are called mixed acid and 2-3 butanediole pathway. Mixed acid pathway, glucose ends up as formic acid, acetic acid, lactic acid, succinic acid. The test used to detect this pathway is the Methyl red test.●2-3 butanediol pathway, butanediol and ethanol are the main end products but a small amount of acid produced. The VP test detects this pathway by detecting acetoin which is an intermediate product on the way to butanediol E. coli (+) and Klebseilla (-).

�� Methyl red test Voges-proskauer test� �

��Citrate Test�

• Citrate Test:is used to detect bacteria that have the ability to break down citrate and turn it into citrate and use it as the only carbon source, by the enzyme Citrate aldolase, which is produced by many bacteria. It was found that E.coli (-) and Klebsiella pneumonia (+) use citrate in the culture media called "Simmons Citrate agar" as a source of carbon, containing the reagent Bromothymol blue

Simmon’s citrate agar

Motility test

• Determining the presence or absence of flagella is done by "indirect" methods.

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• We can also utilize tubes of Motility Medium where in only motile bacteria can move away from the line of inoculation in the low (0.5% or less) concentration of agar

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• Motile bacteria is detected by turbidity (cloudiness) in the medium.
• Salmonella spp. Positive (+) for this test ,Shigella spp Negative(-) for this test.

Triple Sugar Iron test (TSI)

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• TSI agar is a medium used in the identification of Gram-negative enteric rods.

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• The medium measures a bacterium's ability to utilize three sugars, glucose, sucrose and lactose, the concentrations of which are 0.1%, 1.0%, and 1.0%, respectively.

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• A pH indicator (phenol red) included in the medium can detect acid production from fermentation of these carbohydrates. A yellow color change indicates acid in the medium , while no color change indicates an alkaline surrounding.

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• TSI agar can also detect H2S production.

Urease test

• Urease is an enzyme that breaks the carbon-nitrogen bond of amides to form carbon dioxide, ammonia, and water.
• Members of genus Proteus are known to produce urease.
• Urease can be detected by plating bacteria onto an amide containing medium, specifically urea.
• When urea is broken down, ammonia is released and the pH of the medium increases (becomes more basic). This pH change is detected by a pH indicator (phenol red) that turns pink in a basic environment. A pink medium indicates a positive test for urease.

Shigella species

• S. dysenteriae
• Biochemical Tests
• TSI K/A with NO gas
• LIA K/A
• Urea –
• Motility -
• All ferment mannitol except S. dysenteriae
• S. sonnei may show delayed lactose fermentation

Salmonella

• Biochemical Tests
• TSI K/A + gas and H₂S: S. typhi produces only a small amount of H₂S and no gas , and S. paratyphi A produces no H₂S
• LIA K/K with H₂S with S. paratyphi A giving K/A results
• Urea –
• Motility +
• Citrate +/-
• Indole -

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Parasitic infections

• Parasites can enter your body through contamination food or water. Parasites that cause diarrhea include. Entamoeba histolytica, and Giardia lamblia
• Entamoeba histolytica(Amebic Dysentery)

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Morphology

• Different form of E. histolytica;

1- mature trophozoite have a

• Single nuclei with an even lining
• Of peripheral chromatin

2-cyst have 4 nuclei

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Trophozoite character

• Size: 12-60^μm in diameter;
• Invasive form (magna) contain RBC, E. histolytica
• Pseudopodia:
• Motility:
• Endoplasm: may be contain ingested RBC
• Nucleoplasm:

Non-invasive form

invasive form

Different form of E.histolytica cyst

Life cycle

Life cycle

Giardiasis

• Giardia intestinalis
• Protozoal parasite
• Also known as:
• Giardia lamblia

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Parasite Stages

• Cyst

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• Trophozoit

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Trophozoite of G.lamblia

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• Cyst Of G.lamblia

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Life Cycle

• A single host: the man.
• Two stages:
• Trophozoite and cyst
• Four nucleated mature cyst is the infective form.
• Trophozoites browse on the mucosal surface by an oval sucker, multiplied asexually by binary fission.

Diagnosis

• Direct observation in feces
• Trophozoites
• “Tear drop” shape
• Two nuclei and tumbling mobility
• Cysts
• Approximately 13 microns long
• Oval, with 2-4 nuclei
• Immunofluorescence
• ELISA, PCR

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Cestodes (Tape Worms)

• Taenia saginata (beef tapeworm)
• Taenia soilum
• In both species the infective stage is the cysticercus.
• Cows are the intermediate host of T. saginata
• Pig are the intermediate host of T. solium

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• Definitive host: Human.
• Diagnosis:
• By finding gravid proglotid or eggs in stool.
• Eggs present in stool less often are proglottids.
• Direct fecal smear.

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Gravid proglottids of (left) Taenia saginata and (right) T. solium. Injection of India ink in the uterus allows visualization of the primary lateral branches. Their number allows differentiation between the two species: T. saginata has 15 - 20 branches on each side, while T. solium has 7 - 13. Note the genital pores in mid-lateral position

Taeniasis:�Diagnosis