Microbiology Laboratory Turkey

Mikrobiyoloji Ile Ilgili Tüm Konuların Kısa ve Öz Anlatımları. Microbiology Lab Information.

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pseudomonas etiketine sahip kayıtlar gösteriliyor. Tüm kayıtları göster
pseudomonas etiketine sahip kayıtlar gösteriliyor. Tüm kayıtları göster

14 Mart 2019 Perşembe

Mart 14, 2019

Oxidation-Fermentation (OF) Test (or Hugh-Leifson Test)

Oxidation-Fermentation (OF) Test (or Hugh-Leifson Test)

The Oxidation-Fermentation (OF) Test tests the metabolism of sugar by prokaryotic cells.  Cells may metabolize sugar in a variety of pathways, and the OF Test is studying whether sugar is metabolized by aerobic respiration or by an anaerobic pathway including fermentation.  The OF medium has a low agar and peptone content, and a high sugar content, making it a semi-solid medium that is unlikely to go alkaline from protein utilization if the sugars are metabolized.  Sugar fermentation produces fairly strong acids while aerobic respiration produces only weak acids, either of which will turn the indicator, Bromthymol Blue, yellow.  Bromthymol Blue is green around a neutral pH and blue at an alkaline pH (>7.5).  

Two tubes are stab inoculated with an organism, then one tube is covered with mineral oil, creating an anaerobic environment to study fermentation.  The other tube is left uncovered with oil, creating an aerobic environment to study aerobic respiration.  Organisms that do not utilize the sugar in both tubes will leave the media blue or green (non-saccharolytic, non-sugar user).  Organisms that only engage in aerobic respiration will turn the media yellow in the top of the uncovered (aerobic) tube while the covered tube remains blue or green.  

Organisms that only slowly ferment the sugars will be slightly yellow at the top of both tubes.  Organisms that slowly ferment the sugars and aerobically respire the sugars will also be slightly yellow at the top of both tubes.  Organisms that strongly ferment the sugars will be yellow throughout both tubes.  Organisms that both stronly ferment and aerobically respire the sugars will also be yellow throughout both tubes.  Obviously, this test can only differentiate organisms into three or four groups:

  1. Non-sugar metabolisers
  2. Aerobically respiring organisms
  3. Organisms that either aerobically respire and ferment or ferment only
  4. And organisms that either aerobically respire and slowly ferment or slow fermenters only
➤The semi-solid medium also allows for the detection of motile organisms which will radiate out from the stab.

Aim of Test: To differentiate bacteria species on their ability of aerobically respire or ferment sugar and to determine motility.  This test allows for the differentiation of the fermenting Enterobacteriaceae from the aerobically respiring Pseudomonas and Bordetella and from the non-sugar using Alcaligenes and Moraxella

Procedure:
  1. Obtain two tubes for each strain being tested (your instructor will inform you of which strains to test).  
  2. Label the tubes, including your name, the date, and the test name.  
  3. Do NOT use your Wire loop, as this will soon ruin it. Use your inoculating needle for this test.
  4. If assaying for motility, care must be taken with the stab.  Be certain that your needle is straight and that your stab is straight down and then pulled up exactly the way it went down.
  5. Aseptically transfer some bacteria to the inoculating needle.  This is done by flaming your needle, COOLING IT WELL, picking up bacteria on the needle by rolling the tip in a colony of bacteria (oe scaping it).  Sometimes students get no growth in this test from just briefly cooling the tip of the needle: the heat from above the tip moves down the needle and kills the bacteria.  So coool it well.
  6. Stab the needle straight down into the tube going about 2/3 of the way down and then pulling the needle straight out the way you came in.
  7. A poor stab will make the motility reading difficult, so repeat the test if needed.
  8. Repeat this procedure for the second tube of the set.
  9. Overlay one tube of each set with a thin layer of sterile mineral oil (about 4 mm).
  10. Remind your instructor to set up a control tube (an uninoculated control).  Gently place your tubes in the rack where instructed.  Rough handling may distort the test.
  11. Incubate the tubes at room temperature for about 5 days (or at 35-37 C for 2 days).
Results:
  1. Examine the tubes for color changes, noting where the color has changed.  If tubes have no visible growth and have not changed color or the organism is slow growing, additional incubation time may be required.
  2. Look for motility as in the motility test.
  3. Check your results against the table below.
Note to the Instructor: 
Although at first glance this test looks pretty interesting, it actually adds little but confirmation to the knowledge obtained from the Utilization of Glucose Test and the Thioglycollate Test. If given a choice between the two, the latter tests are recommended instead of the OF Test.

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25 Ekim 2018 Perşembe

Ekim 25, 2018

PSEUDOMONAS (TÜRKÇE)

PSEUDOMONAS

Pseudomonas aeruginosa hareketli, sporsuz, kapsülsüz, gram (-) negatif basildir. Bazen çift çift, bazen de kısa zincirler halinde görünür. Çoğu kez bir ucunda tek, nadiren 2-3 kirpiği vardır ve çok hareketlidir. 

 Üreme Özellikleri  
Pseudomonas aeruginosa, aerob olmakla beraber anaerob üreyebilen türlerine de rastlanır. Optimal üreme ısısı 30-37oC‟dir. 42oC'de üreyebilmesi Pseudomonas aeruginosa için önemli bir özelliktir. Optimal pH'sı 7,2'dir. Hafif alkali ortamda bol ürer, kuruluğa fazla dirençli değildir; nemli güneşten uzak yerlerde, toprakta, yüzeysel sularda, yiyeceklerde, hastanelerde zemin ve aletler üzerine yerleşerek  uzun süre canlı kalır. Fırsatçı patojen olarak çeşitli hastalıklara yol açar. 

Adi besiyerinde üreyebilir; buyyonda yüzeyde zar yaparak bol ve homojen bir üreme gösterir; zarın hemen altında mavi-yeşil pigment oluşturur. Peptonlu suda da aynı şekilde ürer. Kanlı agarda hemoliz yapar. Hemoglobini tam olarak hemoliz ettiğinden kanlı agar besiyerindeki kolonilerin etrafında temiz ve berrak zon oluşturur. Pseudomonas aeruginosa kültürlerinde; inci beyazı koloni görüntüsü, tatlımsı aromatik meyve, menekşe, sabun gibi güzel kokusuyla tanınır. Pseudomonas aeruginosa piyosiyanin {mavi-yeşil), piyorubin (kırmızı-kahverengi) ve flöressein (yeşil-sarı ve flöresan) gibi pigmentler üretir. Flöresseini tüm Pseudomas türleri oluşturabilirken piyosiyanini, sadece Pseudomonas aeruginosa oluşturabilir. Bakterinin kesin tanısı, 42°C de üreme yetisi ve pigment üretiminin incelenmesiyle konur. Aerobik olmaları nedeni ile gıdaların yüzeyinde hızlı gelişebilmeleri sonucu okside ürünler ve mukoz madde oluşturur. Süt içinde de iyi ürer, sütün pıhtılaşmasında ve çıkardığı pigmentten dolayı sarı-yeşil renk almasına neden olur, laktoz (), H2S ve indol yapmaz, glikozda asit yapar, gaz yapmaz. 

 Antijenik Özellikleri 
Pseudomonas aeruginosa'da, O antijeni bulunur. Ayrıca H ve pilus antijenleri de saklanmıştır.  

Yaptığı Hastalıklar 
 Yara ve yanık enfeksiyonları, idrar yolu enfeksiyonları, menenjit, göz enfeksiyonları, bronşit ve bronkopnömoni, septisemi.    
GRAM STAINING


Laboratuvar Teşhisi   
İnceleme örnekleri: Kan, idrar, BOS, balgam, cerahat, yara ve yanık sürüntüleri.   
Teşhis Yöntemleri  
➤ Kültür; inceleme örnekleri, buyyon, adi jeloz, kanlı jeloz ve MacConkey gibi besiyerlerine ekilir. Saf kültürü elde edilir. 
➤ Biyokimyasal testler; biyokimyasal özellikleri değerlendirilerek teşhis edilir.  

OKSİDAZ TESTİ İÇİN TIKLAYIN
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Ekim 25, 2018

PSEUDOMONAS

PSEUDOMONAS

Pseudomonas aeruginosa has become an important cause of infection, especially in patients with compromised host defense mechanisms. It is the most common pathogen isolated from patients who have been hospitalized longer than 1 week, and it is a frequent cause of nosocomial infections. Pseudomonal infections are complicated and can be life-threatening.

Signs and Symptoms
Pseudomonal infections can involve the following parts of the body, with corresponding symptoms and signs:
➤ Respiratory tract (eg, pneumonia)
➤ Bloodstream (bacteremia)
➤ Heart (endocarditis)
➤ CNS (eg, meningitis, brain abscess)
➤ Ear (eg, otitis externa and media)
➤ Eye (eg, bacterial keratitis, endophthalmitis)
➤ Bones and joints (eg, osteomyelitis)
➤ GI tract (eg, diarrhea, enteritis, enterocolitis)
➤ Urinary tract
➤ Skin

Physical findings depend on the site and nature of the infection, as follows:
➤ Endocarditis: Fever, murmur, and positive blood culture findings; peripheral stigmata such as Roth spots, Janeway lesions, Osler nodes, splinter hemorrhages, and splenomegaly
➤ Pneumonia: Rales, rhonchi, fever, cyanosis, retractions, and hypoxia; occasionally shock; with cystic fibrosis, clubbing, increased anteroposterior (AP) diameter, and malnutrition
➤ GI tract: Fever, signs of dehydration, abdominal distention, and signs of peritonitis; physical findings of Shanghai fever
➤ Skin and soft tissue infections: Hemorrhagic and necrotic lesions, with surrounding erythema; subcutaneous nodules, deep abscesses, cellulitis, and fasciitis; in burns, black or violaceous discoloration or eschar
➤ Skeletal infections: Local tenderness and a decreased range of motion; neurologic deficits
➤ Eye infections: Lid edema, conjunctival erythema and chemosis, and  evere mucopurulent discharge
➤ Malignant otitis externa: Erythematous, swollen, and inflamed external auditory canal; local lymphadenopathy
➤ Bacteremia: Fever, tachypnea, and tachycardia; hypotension and shock; jaundice 

>
Diagnosis
Laboratory studies that may be helpful include the following:
➨ Complete blood count (CBC)
➨ Blood cultures
➨ In urinary tract infection (UTI), urinalysis
➨ In pneumonia, culture of sputum and respiratory secretions, as well as blood gas analysis 
➨ Wound and burn cultures and cultures from other body fluids and secretions according to the clinical scenario
➨ Gram stain and culture of CSF if meningitis is suspected

➥Pseudomonas aeruginosa is member of the Gamma Proteobacteria class of Bacteria. It is aerobic bacterium belonging to the bacterial family Pseudomonadaceae. 

 GRAM Stain
Pseudomonas aeruginosa is a Gram-negative rod measuring 0.5 to 0.8 µm by 1.5 to 3.0 µm. Almost all strains are motile by means of a single polar flagellum.

Cultivation on Blood Agar
Pseudomonas aeruginosa has very simple nutritional requirements. It is often observed "growing in distilled water", which is evidence of its minimal nutritional needs. In the laboratory, the simplest medium for growth of Pseudomonas aeruginosa consists of acetate as a source of carbon and ammonium sulfate as a source of nitrogen.

P. aeruginosa isolates may produce three colony types. Natural isolates from soil or water typically produce a small, rough colony. Clinical samples, in general, yield one or another of two smooth colony types. One type has a fried-egg appearance which is large, smooth, with flat edges and an elevated appearance. Another type, frequently obtained from respiratory and urinary tract secretions, has a mucoid appearance, which is attributed to the production of alginate slime. The smooth and mucoid colonies are presumed to play a role in colonization and virulence.

The oxidase test is a biochemical reaction that assays for the presence of cytochrome oxidase, an enzyme sometimes called indophenol oxidase. In the presence of an organism that contains the cytochrome oxidase enzyme, the reduced colorless reagent becomes an oxidized colored product. The oxidase test often uses a reagent, tetra-methyl-p-phenylenediamine dihydrochloride (or KOVÁCS reagent), as an artificial electron donor for cytochrome c. When the reagent is oxidized by cytochrome c, it changes from colorless to a dark blue or purple compound, indophenol blue. There are many method variations to the oxidase test. These include, but are not limited to, the filter paper test, filter paper spot test, direct plate method, and test tube method.

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13 Ekim 2018 Cumartesi

Ekim 13, 2018

OXIDASE TEST

OXIDASE TEST PROCEDURE

Oxidase Test
Many organisms that respire have the cytochrome C oxidase enzymes in their electron transport chain that transfers electrons to oxygen, reducing oxygen to either water or hydrogen peroxide.  (If the toxic hydrogen peroxide is generated, catalase will convert the hydrogen peroxide to water and oxygen.)  The tests described here use Kovac's  oxidase reagent (tetramethyl-p-phenylenediamine dihydrochloride) that changes color when it becomes oxidized by cytochrome C oxidase.  As this enzyme requires oxygen, it is present only in aerobes, a few microaerophiles, and facultative anaerobes.  Because all known strains that are oxidase positive are catalase positive (either +i or +s), but not all catalase positive strains are oxidase positive, these two tests together may really help narrow down the identification of an unknown.
➤ This test can be performed using one of two similar procedures, your instructor will inform you of which.

Purpose: 
to test for the presence of cytochrome oxidase.  This test is helpful in identifying members of Neisseria, Enterobacteriaceae, Pseudomonadaceae, to aid in differentiating Gram negative nonenterics from Enterobacteriaceae, and in identifying a few others.

 Colonial Growth Procedure:
➤ Have freshly grown growth in streaks, lines, or colonies on a solid media, such as Nutrient Agar or TSA.  Do not use blood agar plates as blood cells contain oxidase and may give a false positive reading.
➤ Watch the time closely.  Directly place two or three drops of the reagent onto some growth.
➤ Within 20 seconds, observe if the growth under the reagent turns blue.
➤ If the color changes blue within 20 seconds, record the test as positive for the presence of cytochrome C oxidase.
➤ Keep watching for a color change to blue within 30 seconds.  If the color changes to blue within 30 seconds, record the test as potentially positive or inconclusive.  If the color does not change to blue within 30 seconds, record your results as negative for the presence of cytochrome C oxidase.  Important, the reagent may oxidize on its own and turn blue, so a color change after 30 seconds is negative.
➤ Compare your results with both a positive and a negative control.

Oxidase Slide Procedure:
➧This procedure requires BBL's DrySlide TM from Becton Dickinson and Company.  These slides already have the reagent on them.
➧Use freshly grown growth or colonies from a solid media that is not blood agar, such as Nutrient Agar or TSA.  Old growth is not reliable.
➧Transfer a large colony or equivalent amount of cells to the reagent slides using a tool that is not metal. Sterile pipettes and the wooden handles of swab sticks are suggested.  Some use cotton swabs to transfer the cells, but too many cells often stay with the swab, so this is not recommended.  If you are unable to pick up a large colony of cells, an agar plug may be cut out of a spare plate of cells with cleaned instruments, remove and invert the plug with cleaned forceps, and place the cells on the plug directly on the BBL's DrySlide TM.  Do not allow the forceps to touch the slide.  (This procedure works well but will likely contaminate the plate, so make sure it is a spare plate.) 
➧Observe the slides for a color change to blue watching the time closely.
➧If the color changes to blue within 20 seconds, record the test as positive for the presence of cytochrome C oxidase.  If the color becomes blue within 30 seconds, record the test as probably positive.
➧If the color does not change to blue within 30 seconds, record the test as negative for the presence of cytochrome C oxidase.
➧Compare your results with both a positive and a negative control.  Note, many specimens will turn blue after 30 seconds from a side reaction, a few as early as 45 seconds, so watch the time closely and record these as negative.

Instructor Notes:
The reagent can oxidize (go bad) and should be tested with a known positive control.  If the reagent looks bad, do not use unless known controls test correctly.  Escherichia coli is oxidase negative and Bacillus subtilis, Alcaligenes faecalis, and the Pseudomonas species are all positive.  With the DrySlides, some known positive controls may not turn blue within 20 seconds (BD BBL's DrySlide TM Oxidase Product Insert, 2013) but usually do within 30 seconds.  The Colonial Growth procedure does seem to work better than the Slide procedure if fresh reagents and fresh cultures are used.  Not only is the Colonial Growth procedure easier to visibly observe than the Slide procedure, but the color change also occurs earlier and further away from the cutoff time.  Collin et al., 1989, say that a clean platinum wire may be used, but determining a clean from a mostly clean wire and a platinum from non-platinum wire may be too difficult for most students so that metal instruments are not recommended.  A trace of iron in a metal instrument that touches the reagent slide may result in a false positive result.  The oxidase test should not be performed on cells that have been grown in a medium containing glucose as glucose fermentation inhibits oxidase.  There are also other oxidase tests that use other reagents, but they are less sensitive than the test and reagent discussed here.


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