Etiket Arşivleri: Escherichia coli

Gram Stains of Bacteria

Gram Stains of Bacteria
Escherichia coli,
Klebsiella pneumoniae,
Enterobacter aerogenes,
Staphylococcus aureus,
Staphylococcus epidermis,
Bacillus subtilis,
Bacillus cereus,
Bacillus megaterium,
Bacillus megaterium,
Micrococcus luteus,
Pseudomonas aeruginosa,
Proteus vulgaris,
Mycobacterium smegmatis

Enterohemorrhagic Escherichia Coli Infections

In today’s presentation we will cover information regarding Escherichia coli (E. coli) and its epidemiology. We will also talk about the history of the disease, how it is transmitted, species that it affects (including humans), and clinical and necropsy signs observed. Finally, we will address prevention and control measures for E. coli, as well as actions to take if E. coli is suspected.

[Photo: Scanning Electron Microscopy (SEM) of Escherichia coli organism. Source: CDC Public Health Image Library]

Escherichia coli is a Gram negative rod (bacillus) in the family Enterobacteriaceae. Most E. coli are normal commensals found in the intestinal tract. Enterohemorrhagic Escherichia coli (EHEC) is a subset of pathogenic E. coli that can cause diarrhea or hemorrhagic colitis in humans. Hemorrhagic colitis occasionally progresses to hemolytic uremic syndrome (HUS), an important cause of acute renal failure in children and morbidity and mortality in adults. Pathogenic strains of this organism are distinguished from normal flora by their possession of virulence factors such as exotoxins.

[Photo: Colorized scanning electron micrograph (SEM) depicting Escherichia coli O157:H7. CDC Public Health Image Library]

The specific virulence factors can be used, together with the type of disease, to separate E. coli into pathotypes. Verocytotoxigenic (or verotoxigenic) E. coli (VTEC) produce a toxin that is lethal to cultured African green monkey kidney cells (Vero cells) but not to some other cultured cell types. There are two major families of verocytotoxins, Vt1 and Vt2. A VTEC isolate may produce one or both toxins. Because verocytotoxin is homologous to the shiga toxins of Shigella dysenteriae, VTEC are also called shiga toxin-producing E. coli (STEC). Enterohemorrhagic E. coli are VTEC that possess additional virulence factors. One key characteristic found in EHEC, but not exclusive to these organisms, is the ability to cause attaching and effacing (A/E) lesions on human intestinal epithelium. Some of the genes that are involved in producing A/E lesions can be used, together with the presence of the verocytotoxin, to help identify EHEC.

  1. coli are serotyped based on the O (somatic lipopolysaccharide), H (flagellar) and K (capsular) antigens. Serotypes known to contain EHEC include E. coli O157:H7, the non-motile organism E. coli O157:H-, and members of other serogroups, particularly O26, O103, O111 and O145 but also O91, O104, O113, O117, O118, O121, O128 and others. Serotyping alone is not enough to identify an organism as an EHEC; virulence factors characteristic of these organisms must also be present. E. coli O157:H7 strains are relatively homogeneous, and nearly all of these organisms carry virulence factors associated with hemorrhagic colitis and HUS.

[Photo: Transmission electron micrograph of E. coli O157:H7 showing flagella. CDC Public Health Image Library]

  1. coli O157:H7 was first described in 1982 in four patients with bloody diarrhea. The initial outbreak was associated with two outlets of the same fast-food chain, and illness was linked to undercooked hamburgers. More recently, other sources for E. coli 0157:H7 have been identified, including apple juice and cider; raw vegetables such as lettuce and spinach; raw milk; and processed foods such as salami. Over the years, E. coli O157:H7 has evolved as a major problem for physicians, public health authorities, and the food industry. Source: Centers for Disease Control and Prevention (CDC). Isolation of E. coli O157:H7 from sporadic cases of hemorrhagic colitis–United States. 1982. MMWR Morb Mortal Wkly Rep. 1997 Aug 1;46(30):700-4.

EHEC O157:H7 infections occur worldwide; infections have been reported on every continent except Antarctica. Other EHEC are probably also widely distributed. The importance of some serotypes may vary with the geographic area.

EHEC infections can occur as sporadic cases or in outbreaks. In North America, EHEC O157:H7 infections are most common from summer to autumn. Seasonality might be caused by seasonal shedding patterns in animals, or it could be due to other factors such as eating undercooked meat at summer barbecues. The incidence of EHEC in humans is difficult to determine, because cases of uncomplicated diarrhea may not be tested for these organisms. FoodNet surveillance from 1996 to 2010 showed that O157 infection caused 0.9 illnesses per 100,000; this represents a decrease compared to the period from 1996 to 1998. In clinical cases, the mortality rate varies with the syndrome. Hemorrhagic colitis alone is usually self–limiting, but death is possible. The number of cases that progress to HUS varies with the organism and the outbreak. Approximately 5-10% of patients with hemorrhagic colitis from EHEC O157:H7 usually develop HUS. Complications and fatalities are particularly common among children, the elderly, and those who are immunosuppressed or have debilitating illnesses. HUS is fatal in 3–10% of children and TTP in up to 50% of the elderly.

This image shows the relative rates of laboratory-confirmed infections with Campylobacter, STEC O157, Listeria, Salmonella, and Vibrio, compared with 1996–1998 rates, by year, according to FoodNet surveillance in the U.S. from 1996-2010. For STEC O157, a 44% decrease was observed. Compared with 2006-2008, the incidence was significantly lower for STEC O157 (29% decrease) in 2010.

Source: Centers for Disease Control and Prevention (CDC). Vital signs: incidence and trends of infection with pathogens transmitted commonly through food–foodborne diseases active surveillance network, 10 U.S. sites, 1996-2010. MMWR Morb Mortal Wkly Rep. 2011 Jun 10;60(22):749-55.

Surveys suggest that EHEC O157:H7 is widespread in cattle herds, but the prevalence in individual animals is low. Some studies have found that this organism is more common in cattle during the summer and early autumn. One study reported that the prevalence was higher when it was cooler, but more bacteria were shed in the summer. Other studies have not found seasonal patterns of shedding. Prevalence rates for EHEC O157:H7 among cattle vary from less than 1% to 36%, depending on the country, type of herd studied and other conditions. Recent studies that use sensitive methods for detection report a higher prevalence than early surveys. However, highly sensitive techniques may also overestimate prevalence, as some animals shedding the organism may not be colonized, but only transiently infected by transmission from super-shedders or the environment.

[Photo: Cattle. Source: USDA ARS]

EHEC are transmitted by the fecal–oral route. They can be spread between animals by direct contact or via water troughs, shared feed, contaminated pastures or other environmental sources. Birds and flies are potential vectors. In one experiment, EHEC O157:H7 was transmitted in aerosols when the distance between pigs was at least 10 feet. The organism was thought to have become aerosolized during high pressure washing of pens, but normal feeding and rooting behavior may have also contributed.

[Photo: Cattle at feed bunk. Source: Scott Bauer/USDA ARS]

The reservoir hosts and epidemiology may vary with the organism. Ruminants, particularly cattle and sheep, are the most important reservoir hosts for EHEC O157:H7. A small proportion of the cattle in a herd can be responsible for shedding more than 95% of the organisms. These animals, which are called super-shedders, are colonized at the terminal rectum, and can remain infected much longer than other cattle. Super-shedders might also occur among sheep. Animals that are not normal reservoir hosts for EHEC O157:H7 may serve as secondary reservoirs after contact with ruminants. Person-to-person transmission can contribute to disease spread during outbreaks; however, humans do not appear to be a maintenance host for this organism.

[Photo: Cow. Source: Larry Rana/USDA]

Foodborne outbreaks with EHEC O157:H7 are often caused by eating undercooked or unpasteurized animal products, particularly ground beef but also other meats and sausages, and unpasteurized milk and cheese. Other outbreaks have been linked to alfalfa or radish sprouts, lettuce, spinach and other contaminated vegetables, as well as unpasteurized cider. Irrigation water contaminated with feces is an important source of EHEC O157:H7 on vegetables. This organism can attach to plants, and survives well on the surface of a variety of fruits, vegetables and fresh culinary herbs. Depending on the environmental conditions, small numbers of bacteria left on washed vegetables may multiply significantly over several days. EHEC O157:H7 can be internalized in the tissues of some plants including lettuce, where it may not be susceptible to washing.

[Photos: (Top) Hamburgers. Source: USDA ARS; (Bottom) Leafy greens. Source: Centers for Disease Control and Prevention]

Multistate outbreaks of foodborne E. coli are not uncommon; for example, in recent years, the CDC has investigated food products ranging from ground beef to vegetables to prepared foods (e.g., cookie dough).

Some human cases are caused by exposure to contaminated soil or water. EHEC are usually eliminated by municipal water treatment, but these organisms may occur in private water supplies such as wells. Swimming in contaminated water, especially lakes and streams, has been associated with some infections. Soil contamination has caused outbreaks at campgrounds and other sites, often when the site had been grazed earlier by livestock.

[Photo: Small lake surrounded by farmland. Source: Neil Mitchell/geograph.org.uk (Creative Commons) via http://commons.wikimedia.org/wiki/File:Small_Lake_surrounded_by_farmland_-_geograph.org.uk_-_1752021.jpg]

The incubation period for disease caused by EHEC O157:H7 ranges from one to 16 days. Most infections become apparent after 3-4 days; however, the median incubation period was 8 days in one outbreak at an institution. Person-to-person transmission occurs by the fecal-oral route. Most people shed EHEC O157:H7 for approximately 7 to 9 days; a minority can excrete this organism for 3 weeks or longer after the onset of symptoms. In a few cases, shedding may continue for several months. Young children tend to shed the organism longer than adults. Transmission is particularly common among children still in diapers.

Most symptomatic cases begin with diarrhea. Some cases resolve without treatment in approximately a week; others progress to hemorrhagic colitis within a few days. Hemorrhagic colitis is characterized by diarrhea with profuse, visible blood, accompanied by abdominal tenderness, and in many cases, by severe abdominal cramps. Some patients have a low–grade fever; in others, fever is absent. Nausea and vomiting may be seen, and dehydration is possible. Many cases of hemorrhagic colitis are self–limiting and resolve in approximately a week. Severe colitis may result in intestinal necrosis, perforation or the development of colonic strictures.

Hemolytic uremic syndrome occurs in up to 16% of patients with hemorrhagic colitis. This syndrome is most common in children, the elderly and those who are immunocompromised. It usually develops a week after the diarrhea begins, when the patient is improving. Occasionally, children develop HUS without prodromal diarrhea. HUS is characterized by kidney failure, hemolytic anemia and thrombocytopenia. Extrarenal signs including CNS involvement with lethargy, irritability and seizures are common. In more severe cases, there may be paresis, stroke, cerebral edema or coma. Respiratory complications can include pleural effusion, fluid overload and adult respiratory distress syndrome. The form of HUS usually seen in adults, particularly the elderly, is sometimes called thrombotic thrombocytopenic purpura (TTP). In TTP, there is typically less kidney damage than in children, but neurologic signs including stroke, seizures and CNS deterioration are more common. Death occurs most often in cases with serious extrarenal disease such as severe CNS signs. Approximately 65–85% of children recover from HUS without permanent damage; however, long-term renal complications including hypertension, renal insufficiency and end-stage renal failure also occur.

Because humans do not normally carry EHEC, clinical cases can be diagnosed by finding these organisms in fecal samples. Food and environmental samples may also be tested to determine the source of the infection. There is no single technique that can be used to isolate all EHEC serotypes. Selective and differential media have been developed for EHEC O157:H7, including MacConkey agar, hemorrhagic colitis agar, and commercial chromogenic agars. Colonies suspected to be EHEC O157:H7 are confirmed to be E. coli with biochemical tests, and shown to have the O157 somatic antigen and H7 flagellar antigen with immunoassays. A variety of tests including enzyme-linked immunosorbent assays (ELISAs), agglutination, PCR, immunoblotting or Vero cell assay can be used to detect the verocytotoxin or its genes. Phage typing and pulsed field gel electrophoresis can subtype EHEC O157:H7 for epidemiology; these tests are generally done by reference laboratories. Subtyping is important in finding the source of an outbreak and tracing transmission. Serology is also valuable in humans, particularly later in the course of the disease when EHEC are difficult to find.

[Photo: MacConkey agar culture plate with Escherichia coli bacteria. Source: CDC Public Health Image Library]

Treatment of hemorrhagic colitis is supportive, and may include fluids and a bland diet. Antibiotics are controversial and are usually avoided: they do not seem to reduce symptoms, prevent complications or decrease shedding, and they may increase the risk of HUS. The use of antimotility (antidiarrheal) agents in hemorrhagic colitis also seems to increase the risk for developing HUS. Patients with complications may require intensive care including dialysis, transfusion and/or platelet infusion. Patients who develop irreversible kidney failure may need a kidney transplant.

Ruminants, especially cattle and sheep, are the major reservoirs for EHEC 0157:H7. Bison and deer can be infected. This organism can sometimes be found in other mammals including pigs, rabbits, horses, dogs, raccoons and opossums, and in birds including chickens, turkeys, geese, pigeons, gulls, rooks and various other wild birds. In some instances, it is not known whether a species normally serves as a reservoir host or if it is only a temporary carrier. For example, rabbits shedding EHEC O157:H7 have caused outbreaks in humans, but most infected rabbits have been found near farms with infected cattle. The reservoir hosts for non-O157 EHEC are poorly understood.

[Photo: (Top) Cattle. Source: Alice Welch/USDA; (Bottom) Sheep. Source: Danelle Bickett-Weddle/CFSPH]

EHEC O157:H7 has not been associated with illness in naturally infected animals. In experimentally infected calves, this serotype does not seem to cause disease in animals older than one week of age. There is one report of bloody or mucoid diarrhea, with some deaths, after experimental infection of neonatal (less than 2-day-old) calves. Another study reported illness in gnotobiotic piglets. Members of some non-O157 EHEC serogroups including O26, O111, O118 and O103 may cause diarrhea and other gastrointestinal signs in young animals. Subclinically infected animals can shed EHEC. Shedding may be transient or intermittent, and animals that have stopped excreting this organism can be recolonized. Calves are more likely to shed EHEC O157:H7 than adult cattle. Experimentally infected pigs could shed this organism for at least 2 months.

EHEC lesions in ruminants are usually characterized by inflammation of the intestinal mucosa, and are generally limited to the large intestine. In some cases, a fibrinohemorrhagic exudate is present. In rabbits experimentally infected with EHEC O153, the cecum and/or proximal colon were edematous and thickened, and the serosal surfaces had petechial or ecchymotic hemorrhages. Pale kidneys were also reported. Dogs infected with EHEC O157:H7 had no significant gross lesions. In dogs inoculated with a non-O157 EHEC strain, the primary cause of death was microvascular thrombosis leading to kidney failure and multiple organ failure. This syndrome resembled HUS. In these dogs, inflammation and edema occurred in the small and large intestines. The kidneys were pale, with a few petechiae on the serosal surface. The liver was enlarged, with inflammation and necrotic lesions.

[Photo: Hemorrhagic enteritis in canine small intestine due to E. coli O157:H7. Source: Armed Forces Institute of Pathology/CFSPH]

EHEC can be difficult to identify. They are a minor population in the fecal flora of animals. Carrier animals are usually detected by finding EHEC in fecal samples, which are either freshly voided or taken directly from the animal. Rectoanal swabs may also be used in some cases. Selective and differential media have been developed for EHEC O157:H7, including MacConkey agar, hemorrhagic colitis agar, and commercial chromogenic agars. Selective media and isolation techniques have also been developed for few non-O157 EHEC. Immunological and nucleic acid-based tests that detect O and H antigens, verocytotoxin or various genes associated with EHEC can be used for presumptive diagnosis. These rapid tests can determine whether potential pathogens are present in samples before isolation. They include dipstick and membrane technologies, agglutination tests, microplate assays, colony immunoblotting, PCR, immunofluorescence and ELISAs. Although verocytotoxin production can aid identification, VTEC are common in animals, and these organisms are not necessarily EHEC; additional virulence factors must also be identified. Verocytotoxin-negative derivatives of EHEC can occur. The results from rapid tests are confirmed by isolating the organism. Some kits validated for food and meat samples and kits for human clinical samples may lack sensitivity when testing fecal samples from animals. Although cattle can produce antibodies to O157, serology is not used routinely in animals to diagnose infections with VTEC or EHEC.


Gıda Kaynaklı Hastalıklar ( Fırat ÖZEL )

GIDA KAYNAKLI HASTALIKLAR
Fırat ÖZEL, Gıda Mühendisi 2006

Amaç Eğitimin amacı :
Gıda sanayinde hataların sonuçlarını belirtmek. Yaptığımız işin ciddiyetini göstermek.
Dikkatli olunmaması durumunda gıdaların insanların hayatlarını nasıl etkileyeceğini açıklamak.

Gıda Kaynaklı Hastalıklar Gıda kaynaklı hastalıklar :

Çok kişiyi etkileyen salgınlara

Büyük işgücü kayıplarına

Ekonomik kayıplara neden olmaktadır.

Ne yediğimizi düşünüyoruz ?

Gerçekte ne yiyoruz ?

Bizi de etkiler mi ? Gıda kaynaklı hastalıkları genellikle; • Üşütmek, • Mide üşütmesi • Mide bozulması • Yediklerimizin dokunması • Bağırsakların bozulması şeklinde adlandırılır ve dikkate alınmaz.

Gıda Kaynaklı Hastalıklar A.B.D.’de yapılan araştırma sonuçları: 76 milyon gıda kaynaklı hastalık vakası / yıl * ü325 000 hastanelik olay / yıl ü5 000 ölüm / yıl ü7 milyar $ / yıl * A.B.D.’nin nüfusu, 293 milyondur.

Gıda Kaynaklı Hastalıklar Gıdalar aracılığıyla yayılan hastalık ve zehirlenmelere gıda kaynaklı hastalıklar denir. İki grupta incelenir. Gıda kaynaklı hastalıklar Gıda zehirlenmeleri

Gıda Kaynaklı Hastalıklar Patojen mikroorganizma insan vücuduna gıda aracılığıyla giriyor ve faaliyetleri ile hastalık yapıyorsa gıda enfeksiyonu adı verilir. Hastalığın seyri mikrobun türüne göre değişir.

Gıda Zehirlenmeleri Gıda ile taşınan mikroorganizma, insanı salgıladığı bir zehirle etkiliyorsa gıda entoksikasyonu adı verilir. Mikroorganizma gıda üzerinde veya insan vücudunda zehir üretir.

Gıda Kaynaklı Hastalık Nedenleri Hastalık yapan canlılar arasında : Bakteriler Virüsler Parazitler Küfler Mayalar

Mikroorganizmaların Bulaşma Yolları Hayvanlar Toprak Dışkı Bağırsaklar Su Gıda İnsan İnsan

Mikroorganizmalar • Clostridium botulinum • Clostridium perfringens • Escherichia coli • Listeria monocytogenes • Salmonella • Shigella • Staphylococcus aureus

Cl. botulinum E.coli Salmonella L. monocytogenes B. cereus Shigella

Hastalığın Oluşma Şekilleri üYeterli sayıya ulaşmış mikroorganizmanın vücuda alınması ile; ( örn.: Cl. perfringens) – Gıdaya bulaşan mikroorganizmalar uygun şartlarda ürer. – Belli sayıda mikroorganizma barındıran gıda tüketilir.

Hastalığın Oluşma Şekilleri üAz sayıda mikroorganizma barındıran gıda tüketilir ve canlı vücutta çoğalarak hastalık yapar. – Az sayıda mikroorganizma taşıyan gıda tüketilir. – Uygun ortamı bulan mikroorganizma ürer. – Belli sayıya ulaşınca hastalık oluşur.

Hastalığın Oluşma Şekilleri üGıdada üreyen mikroorganizma zehir üretir. ( örn.: S. aureus) – Gıdaya bulaşan mikroorganizma çoğalır. – Belli sayıya ulaşan mikroorganizma zehir üretir. – Zehir içeren gıda tüketilir.

Hastalığın Oluşma Şekilleri üAz sayıda mikroorganizma içeren gıda vücuda alınır ve vücutta zehir üretir. ( örn. : Cl. botulinum) – Az sayıda mikroorganizma gıdaya bulaşır. – Gıdanın tüketilmesi ile vücuda alınır. – Uygun ortam bulan mikroorganizma zehir üretir.
Hastalık ve Zehirlenmeler Botulizm : Mikroorganizma : Clostridium botulinum Gıda : Konserve (sebze,tavuk, balık, et), tütsülenmiş balık, yetersiz işlenmiş düşük asitli gıdalar. Belirtileri : Çift görme, yutkunamama, konuşmakta zorluk, ileri solunum sistemi felci, %20 ölüm oranı. Oluşumu : 12-72 saat Süresi : Birkaç gün ile birkaç ay arası.

Botulizm sonucu

Hastalık ve Zehirlenmeler Cl. Perfringens zehirlenmesi: Mikroorganizma : Clostridium perfringens Gıda : Pişmiş et ve tavuk eti, çorba, sıcak ortamda birkaç saat bekleyen sos ve et suları. Belirtileri : İshal, karın ağrısı, gaz. Oluşumu : 8-16 saat Süresi : 24-48 saat

Hastalık ve Zehirlenmeler E. coli Enfeksiyonları : Mikroorganizma : Escherichia coli Gıda : Az pişmiş et, çiğ süt, çiğ meyve suları, taze meyve ve sebze. Belirtileri : İleri ishal çoğunlukla kanlı, karın ağrısı, kusma, az ateş. Oluşumu : 1-8 gün Süresi : 5-10 gün

Hastalık ve Zehirlenmeler Listeriosis: Mikroorganizma : Listeria monocytogenes Gıda : Çiğ süt, yumuşak peynir, az pişmiş et, tavuk eti. Belirtileri : Baş ağrısı, ateş, ishal. Oluşumu : 9-18 saat bağırsak; 2-6 hafta menenjit. Süresi : Değişir.

Hastalık ve Zehirlenmeler Salmonellosis : Mikroorganizma : Salmonella spp.(2300 çeşit) Gıda : Çiğ ve az pişmiş et ve tavuk eti, yumurta, patörize olmayan süt ürünleri. Belirtileri : İshal, ateş, karın krampları, kusma. İleri vakalar yüksek ateşe bağlı ölüm. Oluşumu : 1-5 gün Süresi : 2-7 gün

Hastalık ve Zehirlenmeler Staphylococcal zehirlenme : Mikroorganizma : Staphylococcus aureus Gıda : Krema, yumurtalı salata, patates salatası, salam, jambon, peynir. Belirtileri : ani bulantı ve kusma, karın ağrısı, ishal ve ateş de oluşabilir. Oluşumu : 1-6 saat Süresi : 24-48 saat

Fırat ÖZEL 0 507 454 33 57 firatozel@gmail.com firatozel.wordpress.com 27

Differential Staining Gram Staining, Endospore Staining & Capsule Staining

Purpose:

To learn what is differential staining and its purposes
To perform three types of differential staining: Gram staining, Endospore staining and capsule staining
To understand their value when used to stain clinical specimen
To understand the different characteristics of bacteria and thus the differentiation in staining
To understand the basic characteristic of Gram (+) and Gram(-) bacteria and distinguish between them
To be familiar with the importance of endospores and capsule in bacteria
To have a background of the mechanisms of the dyes used in microbiological studies.
To learn dyes used at different staining methods in differential staining

Procedure:

A) Gram Staining
In this procedure different types of bacteria are used throughout the laboratory. These are:

Staphylococcus aureus,
Enterobacter aerogenes,
Bacillus substilis,
Salmonella anatum,
Escherichia coli,
Proteus vulgaris.

Our group (#7) used two of them: Escherichia coli and Proteus vulgaris. These were overnight cultures.
Remove a loopful of NB which contain E.coli and P.vulgaris and placed it on different slides separately and allow it to air dry.
Dry smear, and cover the bacteria on the slid with crystal violet and wait for 1 min.
Pour crystal violet and rinse gently with distilled water and then cover the smear with iodine for 1 min. Then pour off the dye and rinse gently with distilled water.
Allow 95% ethanol to flow over the smear using dropper. The crystal violet will wash off the smear, so we stopped washing till the smear is colorless. (It is important not to over decolorize otherwise Gram (+)’s will lose the violet stain. Then we immediately rinsed with distilled water)
Cover the smear with safranin and wait for 1 min and then wash with water and blot carefully with tissue paper.
Finally after the slide is air-dried, then observe the bacterial cells under 40X and 100X, pay attention to the cell size, shape and color.

B) Endospore staining
In this part of the experiment we stained the endospores of Bacillus substilis with Malachite green, (an old culture, kept for 3 days at 370C)

Prepare a smear of B. Subtilis, and since the dye used is carcinogenic deal the slides with gloves
Put the slide to tripod places in staining rack, and cover the smear with Malachite green and wait for 1 min
Passed the flame under the slide for many times still vapor was observed but not boiling
Cooled the slide and washed it with water, and then we apply safranin and wait for 1 min. After 1 min has passed rinse the slide with water and performe blotting
Finally observe slides under 40X and 100X

C) Capsule staining: use Klebsiella pneumoniae species to see capsule formation but be careful about pathogenecity of bacteria.

Put a drop of Indian ink to one edge of the slide, and after we obtain aseptically a loop of Klebsiella pneumonia culture and mixed with the ink.
Spread the ink with a second slide and apply it for air dry. And finally cover it with safranin for 1 min.
Finally rinse the dye with water and blot the slide and observe it under 100X.

Results:

Species
Observed colors
Expected colors
S.aureus
purple
purple
E.coli
pink/pink
pink
S.anatum
purple
pink
P.vulgaris
purple
pink
B.subtilis
pink/pink/pink
purple
E.aerogenes
pink/pink
pink

Species
Gram Staining(Expected)
S.aureus
positive(+)
E.coli
negative(-)
S.anatum
negative(-)
P.vulgaris
negative(-)
B.subtilis
positive(+)
E.aerogenes
negative(-)

Our group observed two bacteria for gram staining. First of Escherichia coli was rod shaped, pink colored. Due to pink color of it we conclude that it is Gram (-),d does not have any cell wall but have outer membrane. However, Proteus vulgaris was coccus and color after staining was purple so that we understand that it is Gram (+), it has cell wall. Expected result is also same with experimental results of our group but it is not true for B.subtilis observations. They saw pink color this may be due to high staining of Crystal violet and cause of degrade cell wall properties, and also reason for wrong observation might be that they used clear background and high amount of light so they thought that it is pink rather than purple. It is also possible some genetic problems or degradation or loss of peptidoglaycan at their cell wall so they can not show Gram (+).

For the endospore staining we used Bacillus subtilis and the result was failure because Bacillus subtilis is very small to see even at 100X oil immersion. However we know that cell having blackish dots one side of the cells should be endospore part of endospore formed bacteria and also vegetative part should be stain pink due to safranin.

For the capsule staining we observed shining part of capsule with careful observation but it is not certain that we saw correct thing because we did not use India ink and so that we could not stain surface with black effectively.

Discussion:

In this experiment, we have learned several basic molecular biology techniques and use of equipments such as differential staining techniques such as Gram staining, endospore staining and capsule staining.

Stains are chemical substances that make bacterial cells more visible by increasing the contrast between the cell and background. The dyes are usually organic molecules of complex. All dyes selectively absorb light of certain wavelengths and thus have a color. Many dyes useful for staining bacterial cells also specifically bind to the surface of bacterial cells. This category of dye is referred as positive stains. Some dyes do not bind to the surface of bacterial cells. These dyes called negative stains, they stain environment like Indian ink staining. With negative stains bacterial cells appear white. We use staining as help to provide information about cell morphology- size, shape and arrangement. In the case of Gram staining, it can also provide more detailed information about such cell propertied as the presence of a cell wall.

Gram staining is one of the examples of positive staining. Crystal violet (a positively charged) evidently binds to a negatively charged molecule, probably in the cell envelope or nucleic acid in the center of cell. Iodine must be added to form insoluble iodine-CV complexes. EtOH is used as a mordant, the Gram (+) bacteria with their thick peptidoglycan layers and with relatively little lipid decolorize slowly whereas the Gram (-) bacteria decolorize rapidly because of the ethanol dissolves their outer membrane lipid allowing the crystal violet-iodine complex to be released or because their thin peptidoglycan layer can not trap the complex. Finally, counterstaining with the safranin is done to make the Gram (-) cells visible in the microscope.

For our experiment we used S.aureus, E.coli, S.anatum, P.vulgaris, B.subtilis, E.aerogenes cultures. Most of the results are expected but there are also some unexpected results which most probably caused by some errors during staining and observation. Becasue purple color and pink color are very similar; we might see wrong color under microscope.

Another stain that has been used for years is the indirect or negative stain. The advantage of this stain is that it is the simplest and often quickest means of discovering cell shape and possibly refractive inclusions and endospores. It also does not distort bacteria, which may happen with Gram stains, since cells sometimes, shrink as a result of heat fixation. We used Schaffer-Fulton Technique for endospore staining of Bacillus substilis culture. Malachite Green is used as a primary stain then we apply steam to enhance penetration of the dye. Then we cooled and decolorize the slides by rinsing with water. Then we apply safranin as a counterstain to stain the colorless vegetative parts. At the end of the process spores appear in green and vegetative parts appear in red. Actually, we saw endospore parts black in general and vegetative part as red.

Principles of negative staining are as followings in case of capsule staining technique. We used a polysaccharide-containing material, in other words glycocalyx, lying outside of the bacterial cell to stain. It is related with pathogenecity because it aids attachments, contributes to immune system and it prevents dessication by binding some water. Capsules are largely water soluble and nonionic so they don’t bind to ordinary stains. Also heat activation can not used because it damages the capsule. So we used Indian ink for the background staining and safranin for staining the cell. This is actually not Indian ink but we could not find any Indian ink so that we tried to use usual, custom ink to stick cells to surface and stain surface black. As a result, we could not observe the capsule. Most probably the dye we used did not work effectively as effective as Indian ink.

In conclusion, these exercises were successful and they were a good and essential part of study and learn microbiology. New procedures were practiced, and further understanding of differential stainings, charachters of endospore, capsule, gram staining and principles of stainigs were gained.

REFERENCES:

TAXONOMY, Classification, Nomenclature, Laboratory Identification. Retrieved November 1, 2005 fromhttp://medic.med.uth.tmc.edu/path/00001458.htm
Staphylococcus. Retrieved November 1, 2005 from http://textbookofbacteriology.net/staph.html
Bacillus subtilis. . Retrieved November 1, 2005 from http://en.wikipedia.org/wiki/Bacillus_subtilis.

Escherichia Coli ( İlkay ÜNAL )

Genelde E. coli kısaltması ile veya koli basili olarak bilinen Escherichia coli (okunuşu Eşerişiya koli), memeli hayvanların kalın bağırsağında yaşayan faydalı bakteri türlerinden biridir. E. coli, pediyatrist ve bakteriyolog olan Theodor Escherich tarafından bebek dışkılarında keşfedilmiştir ve adını ondan alır; coli, “kalın bağırsaktan” demektir. E. coli, genel olarak bakteri biyolojisinin anlaşılması amacıyla üzerinde sıkça çalışılmış bir model organizma olmuştur. Canlılar arasında hakkında en fazla şey bilinen organizma olduğu söylenebilir

Başlangıçta bağırsak florası olarak kabul edilen ve burada B vitamini sentezine katılması ile yararlı olarak nitelendirilen E.coli, daha sonraları diyarejenik suşlarının ortaya konulması ile bu bakteriye bakış değişmiştir. Bugün için gıda kaynaklı en tehlikeli patojen bakteri E.coli’nin özel bir serotipi olan O157:H7’dir. } İnsanın bir günde dışkı yoluyla vücudundan geçen E. coli bakteri sayısı 100 milyar ila 10 trilyon arasındadır. Dışkıyı oluşturan bakteriler başlıca anerobik bakterilerdir. Başka hayvanlarda etkisiz olan bazı E. coli tipleri insana bulaştıklarında hastalık yapabilirler. Bunların en ünlüsü sayılan O157:H7 adlı serotip kanlı ishale ve ölüme yol açabilir.

E.coli yaklaşık olarak 2-6 boyunda µm, 0.6- 1.5 µm eninde,düz uçları yuvarlak bakterilerdir. Çoğu hareketli olmakla birlikte hareketsiz olan suşlarıda vardır. Bazı şusları kapsüllüdür. Spor oluşturmazlar. Gram negatiftirler.

Fakültatif anaerop bakteridir } Kan,serum,asit sıvısı ve glukoz ilave edilmeyen besi yerlerinde kolaylıkla üreyebilirler. Optimal üreme ısısı 37 °C optimal pH 7-7.2 dir. Ancak 22-44 °C ve pH 5-8 arasında ürerler. Özellikle 44 °C ‘de üreyebilmeleri benzer bazı bakterilerden ayırt edici özelliktir. } Kanlı agarda hafif nemli görünümlü, 1-2 mm çapında gri renkli koloniler oluştururlar. Etken uzun süreli soğuk ve donmuş muhafaza ortamında canlılığını korur.

E. Coli’nin somatik(O), kirpik (H), kapsül(K) antijeni vardır.antijen yapılarının belirlenmesi özellikle epidemiyolojik çalışmalarda yararlıdır. } O ANTİJENİ; ısıya ve alkole dayanıklıdır } H ANTİJENİ; protein yapıdadır.ısıya, alkole ve proteolitik enzimlere dayanıksızdır. } K ANTİJENİ; kapsul antijenidir.bir çok E.coli suşunda bulunur.üç tiptir.

E. coli ‘nin büyümesi ve bölünmesi. E. coli, orta kısmından yeni hücre duvarı yaparak büyür. Mavi ve kırmızı, sırasıyla, eski ve yeni hücre duvarını gösterir

Bağırsak florasının normal bir üyesi olan E. coli ile konak organizma arasında uyumlu bir ilişki olduğundan bakteri normalde hastalık yapmaz. Ancak, ortama geçmesi halinde, ki bu aynı organizmada başka bir organ olabilir (idrar yolu enfeksiyonu ile mesaneye geçmek gibi) veya başka bir konak organizmanın bağırsağı olabilir, E. coli bir hastalık etmeni olabilir. } Bazı E. coli tipleri içinde bulundukları hayvan için zararsız olmalarına rağmen insana geçtiklerinde hastalık yapabilirler. } Bu hastalıklar arasında başlıca ishalli hastalıklar olmakla beraber idrar yolu enfeksiyonları, menenjit, septisemi ve gram-negatif pnömoni de sayılabilir.

E. coli ‘nin, tavuk, dana ve başka hayvanlarda da hastalık yapabildiği gösterilmiştir. } E. coli içinde hastalık yapan pek çok tipi vardır. Bunlar hasta ettikleri dokular ve hastalık mekanizmalarına bağlı olarak aşağıdaki gibi gruplandırılırlar. } 1)İshalli hastalıklar } 2)İdrar yolu enfeksiyonu

İshalli hastalıklara neden olan E. coli tipleri aşağıdaki gruplara ayrılırlar; } Enterotoksijen E. coli (ETEC) tipleri, enterotoksin üreterek hastalık yapar. Çeşitli toksinler vardır, bazıları bağırsak mukozasına zarar veren sitotoksik enterotoksinlerdir, bazıları bağırsak hücrelerinin su ve elektrolit salgılamalarına neden olan sitotonik enterotoksinlerdir. } insan, domuz, koyun, keçi, sığır, köpek ve atlarda ishal (ateş olmadan) etkeni } ETEC çocuklarda ishale neden olan dünyanın önde gelen bakteriden biridir.

Enteroinvazif E. coli } Sadece insanlarda görülür. } (EIEC) tipleri, doku hücrelerinin içine girip çoğalırlar. Bunun yol açtığı enflamasyon tepkisi doku hasarını artırır. } Yüksek ateş ve ishale sebep olur.

Enteropatojenik E. coli (EPEC) } tipleri dokuya sıkıca bağlandıktan sonra bir enflamasyon reaksiyonu oluştururlar. Toksin salgılayarak değil, hücre içi sinyalizasyona etki ettikleri için ishale yol açtıkları düşünülmektedir. } insanlar, tavşanlar, kediler, köpekler ve atlar içinde ishal etkeni .

Enterohemorajik E. coli (EHEC) Bu grupta olanlar Enteropatojenik özellikler taşımaya ilaveten Şiga toksinleri salgılar. Bu gruba ait olanların en ünlüsü E. coli O157:H7’nin yol açtığı hastalık hemorajik kolit olarak adlandırılır. İshal az sulu, bol kanlı ve mukuslu olur. Insan, sığır ve keçiler bulunur. } EnteroAggregatif E. coli (EAEC), Bağırsak epiteline bağlanıp tuğla gibi dizilmiş bakteriler şeklinde görünür. Bu gruba has bakterilerin salgıladığı toksinler mukozaya zarar verip kronik ishale yol açarlar. Sadece insanlarda görülür.

Diffusely Adherent E. coli (DAEC), Bir yaştan küçük çocuklarda ishale yol açar. Özelleşmiş fimbiralar sayesinde seyrek bir şekilde epitele bağlanırlar ve hücre içi sinyal mekanizmasını etkinleştiriler. Bu grup hakkında az şey bilinmektedir

Uropatojenik E. coli (UPEC) İdrar yolu enfeksiyonlarının %90’ının nedenidir. Bu E. coli tipleri idrar yolu epitel hücrelerine özellikle bağlanabilen fimbriumlara sahiptir. Kadınların üretrasının erkeklerinkinden daha kısa olması nedeniyle bu enfeksiyon kadınlarda daha sıkça görülür. } Dışkıdan gelen bakteri genelde cinsel ilişki sonucu idrar yoluna girer, bakteriler üretrayı tırmanıp mesaneye ulaşırlar. Mesane enfeksiyonuna sistit denir, tedavi edilmediğinde böbrek yangısına (piyelonefrit) dönüşebilir.

E. coli türü içinde büyük bir çeşitlilik vardır, hatta modern tekniklerle gösterilmiştir ki Shigella ve Salmonella familyasının üyeleri aslında E. coli’nin alt- tipleridir. } E. coli türü içinde farklı özelliklere sahip olan, “suş” olarak adlandırılan çeşitli tipler vardır. Bunları birbirinden farklı kılan küçük mutasyonlar olabileceği gibi bütün bir genin, hatta pek çok genin, varlığı veya yokluğu, olabilir. } Suşları farklı kılan genler arasında toksin ve yapışma (adezyon) faktörleri gibi hastalık (virülans) faktörleri vardır. Örneğin O157:H7 suşunun taşıdığı Şiga toksini geni, E. coli ‘ye Shigella’dan geçmiştir.

Aşağıda E. coli ‘nin hastalık yapmasını sağlayan özelliklerin bazıları sıralanmıştır. Bunların hepsi bir arada olmaz, belli E. coli suşları bu faktörlerin belli kombinasyonlarına sahiptir. } Pilus veya fimbriumlar bakterinin üstünde bulunan ve saç görünümlü yapılardır. } Zararsız E. coli ‘ler de piluslara sahip olmakla beraber ETEC tiplerinde bulunan özelleşmiş piluslar onların ince bağırsak epitel hücrelerine bağlanmalarını sağlar. Bu sayede bakteri dışkıyla atılmayıp ince bağırsakta yerleşir ve orada çoğalabilir. } Bu faktörler konak organizmaya özgün olup bakterinin hangi hayvanlarda çoğalabileceğini belirler. Başka tip piluslar idrar yolu hücrelerine veya mesane hücrelerine bağlanmayı sağlarlar, bu yüzden idrar yolu enfeksiyonlarına yol açarlar.

Eksotoksinler. ETEC tiplerinin neden olduğu ishali meydana getiren ST eksotoksini epitel hücrelerinin su emmelerini engeller, LT eksotoksini ise hücrelerin su ve elektrolit salgılamalarına neden olur. EHEC tipi bakteriler ST ve LT eksotoksinleri yoktur, bunlar Şiga toksini salgılarlar, bu toksin bağırsak epitel hücrelerinin ölümüne yol açar, bu yüzden bağırsak su emme yeteneğini kaybeder, sonuç kanlı bir ishaldir } Kapsül hücrenin dışında ek bir koruyucu tabakadır, vücudun koruma mekanizmalarının bakteriyi tanımasını ve imha etmesini engeller

Hemolizin alyuvarların parçanmasını sağlar, salınan demir bakteri için bir besin kaynağıdır. } Sideroforlar kandaki demiri toplamaya yarar. Kanamalı ishalde ve sistemik enfeksiyonlarda bakterinin büyümek için ihtiyaç duyduğu demiri sağlar. } K1 antijeni bakterinin fagositozuna engel olur. } Endotoksin hücre zarında bulunan bir glikolipittir, vücudun ona karşı gösterdiği kuvvetli tepki enflamasyonda önemli rol oynar.

Gıdaların yıkanması patojen E. coli enfeksiyonun yeme yoluyla yayılmasını engellemenin en etkili yoludur. E. coli bulaşmış yiyeceklerin kaynatılması da etkilidir. } Sıcaklığa direnç göstermez. pastörizasyon veya kaynatma ile ölür. (D60 ºC)

Uygun tedavi, enfeksiyonun nedeni olan E. coli tipinin antibiyotik duyarlılığına bağlıdır. } E. coli ‘nin neden olduğu her hastalık için her antibiyotik uygun olmayabilir, bu konuda bir doktora danışmak gereklidir.

hazırlayan İLKAY ÜNAL TEŞEKKÜRLER!..