peptidoglycan
Ocak 31, 2019
Gram Negative Bacteria
Gram Negative Bacteria
Gram negative bacteria are bacteria that do not retain the crystal violet dye in the Gram stain protocol. Gram negative bacteria will thus appear red or pink following a Gram stain procedure due to the effects of the counterstain (for example safranin).
The Gram Stain
In microbiology, the visualization of bacteria at the microscopic level is facilitated by the use of stains, which react with components present in some cells but not others. This technique is used to classify bacteria as either Gram-positive or Gram-negative depending on their colour following a specific staining procedure originally developed by Hans Christian Gram. Gram-positive bacteria appear dark blue or violet due to the crystal violet stain following the Gram stain procedure; Gram-negative bacteria, which cannot retain the crystal violet stain, appear red or pink due to the counterstain (usually safranin).
The reason bacteria are either Gram-positive or Gram-negative is due to the structure of their cell envelope. (The cell envelope is defined as the cell membrane and cell wall plus an outer membrane, if one is present.) Gram-positive bacteria, for example, retain the crystal violet due to the amount of peptidoglycan in the cell wall. It can be said therefore that the Gram-stain procedure separates bacteria into two broad categories based on structural differences in the cell envelope.
Cell Envelope of Gram Negative Bacteria
The Gram negative cell envelope contains an additional outer membrane composed by phospholipids and lipopolysaccharides which face the external environment. The highly charged nature of lipopolysaccharides confer an overall negative charge to the Gram negative cell wall. The chemical structure of the outer membrane lipopolysaccharides is often unique to specific bacterial strains (i.e. sub-species) and is responsible for many of the antigenic properties of these strains. Many species of Gram-negative bacteria are pathogenic. This pathogenicity is often associated with the lipopolysaccharide (LPS) layer of the Gram-negative cell envelope.
Characteristics of Gram Negative Bacteria
Gram-negative bacteria have a characteristic cell envelope structure very different from Gram-positive bacteria. Gram-negative bacteria have a cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane containing lipopolysaccharide. There is a space between the cytoplasmic membrane and the outer membrane called the periplasmic space or periplasm. The periplasmic space contains the loose network of peptidoglycan chains referred to as the peptidoglycan layer.
- Acinetobacter
- Actinobacillus
- Bordetella
- Brucella
- Campylobacter
- Cyanobacteria
- Enterobacter
- Erwinia
- Escherichia coli
- Franciscella
- Helicobacter
- Hemophilus
- Klebsiella
- Legionella
- Moraxella
- Neisseria
- Pasteurella
- Proteus
- Pseudomonas
- Salmonella
- Serratia
- Shigella
- Treponema
- Vibrio
- Yersinia
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13 Kasım 2018 Salı
mikrobiyoloji
Kasım 13, 2018
ENDO Agar
ENDO AGAR
There are actually two similar Endo agars, Endo agar and LES Endo agar. They differ mainly in that LES Endo agar has added nutrients that support a wider growth of strains, but otherwise display similar results. They are both Selective & Differential Media. The selective and differential aspects are due to sodium sulfte, basic fuchsin, and lactose in each medium.
They are Selective because they encourage some bacteria to grow while inhibiting others. Sodium sulfte and basic fuchsin generally inhibits Gram positive bacteria from growing (a few will grow) and generally allowing Gram negative organism to grow (some will not grow).
- If good growth, generally you have a Gram negative bacteria.
- If no growth or if very poor growth, you most likely have a Gram positive.
Both media are Differential because if lactose is utilized, it will create the intermediate acetaldehyde which reacts with the sodium sulfte and basic fuchsin will color the colonies pink/red from the acids produced during lactose fermentation. These plates differentiate between species capable of utilizing lactose and those unable to metabolize it.
- If growth is colorless, beige or not pink, lactose is not utilized and it is probably a Gram negative noncoliform.
- If good growth that is pink/red, lactose is utilized and the bacteria is probably a Gram negative coliform.
- If good pink/red growth that has a bright metallic sheen, lactose is highly utilized and the bacteria is probably a Gram negative coliform.
- (If poor or no growth, recall it is probably a Gram positive organism.)
Purpose: to select for and isolate Gram negative organisms, to check for the presence of coliforms, and to differentiate among the family of Enterobacteriaceae. Its main use is to detect for fecal contamination especially of water and dairy products. Both the Gram negative selection and the detection of coliforms is imperfect, a small percentage of strains do not act as expected.
Procedure:
- Appropriately label you plates. Mark the dish bottom into thirds and label what species will be in each section. It is best to label the side of the bottom plate or write on the bottom in tiny letters so that you will be able to observe the growth clearly.
- Inoculate one third with your unknown streak for isolated colonies if possible. Isolated colonies work best for this test but it is not essential.
- Inoculate one third with E.coli, and the other third with Staphylococcus epidermidis. Generally it is best to keep E.coli well away from the other cultures because it may overgrow them.
- You will want to compare the growth on these plates to growth inoculated on a general purpose media such as NA. or TSA.
- Incubate your plates upside down in the incubator at 35-37 C for 1-2 days.
- Slow growing species may require a day or two of additional growth.
Results
Compare your growth on these plates to growth on a general purpose media. (If no growth on the general purpose media, discard your results and repeat the test.) Look for the presence or absence of growth and if there is growth if it is reduced from normal. It there is growth, check to see if the growth is pink or red in color. One predicts that if an organism grows well on Endo Agar, it is most likely Gram negative, otherwise it is most likely Gram positive. And if the growth is good and pink/red, it is most likely a coliform, otherwise it is likely a noncoliform. Also look if the colonies have a metallic sheen, if they do the organism highly utilizes lactose. Incidentally, a major difference between a coliform and a noncoliform in the family Enterobacteriaceae is simply if it can metabolize lactose.
Notes:
Should confirm their tentative conclusions from this test by performing a Gram stain and testing for lactose utilization. Other species of bacteria may be used as positive and negative controls. E.coli will have good pink/red growth with a metallic sheen, Enterbacter aerogenes will have good pink growth, and S.epidermidis will grow poorly if at all. Instructors may wish to have students test several selective and differential media at the same time and then compare them all to growth on one general purpose media plate to save on the cost.
9 Kasım 2018 Cuma
mikrobiyoloji
Kasım 09, 2018
Eijkman Test
Eijkman Test
Coliform organism is a term used to designate the lactose-fermenting Enterobacteria such as Escherichia coli and Enterobacter. Enterobacteriaceae forms a large group of gram-negative bacteria that inhabit intestinal tract of warmblooded animals. Therefore they constitute the major microbial flora of human faeces. Since coliforms are readily isolated and identified, they are used as indicator organisms to check faecal contamination of food, water and other samples. E.coli is one of the common organisms involved in gram-negative sepsis and endotoxin-induced shock. Eijkman described a method for selective isolation of E.coli from faeces of warm-blooded and cold-blooded animals.
This method had limitations due to the inability to obtain growth after subculturing from positive tubes incubated at 46°C, as acidity and high temperature resulted in death of the culture within 24-48 hours. Perry and Hajna modified Eijkmans original method by decreasing carbohydrate content and adding a phosphate buffer enabling to subculture E.coli after incubation at 46°C for 96 hours or longer where pH was 5.6 unlike 4.5 of Eijkman Medium. Perry modified Eijkman Medium using lactose for isolation of E.coli. This medium can also be used for bacteriological examination in water filtration control work.
Tryptose and lactose in the medium are the energy and the carbon sources respectively. E.coli ferment lactose to form acid and gas. The gas produced gets trapped in the form of gas bubbles in the inverted Durhams tubes. Phosphates buffer the medium whereas sodium chloride helps to maintain the osmotic equilibrium of the medium.
Quality Control
Appearance
Cream to yellow homogeneous free flowing powder
Colour and Clarity of prepared medium
Light yellow coloured, clear solution without any precipitate
Reaction
Reaction of 2.85% w/v aqueous solution at 25°C. pH : 6.8±0.2
pH
6.60-7.00
Cultural Response
Cultural characteristics observed after an incubation at 45.5 to 46ºC for 24 - 48 hours.
Organism Inoculum Growth Gas
(CFU)
Escherichia coli 50-100 luxuriant positive
reaction
Enterobacter aerogenes 50-100 poor negative
reaction
7 Kasım 2018 Çarşamba
mikrobiyoloji
Kasım 07, 2018
Eijkman Testi
Eijkman Testi
29 Ekim 2018 Pazartesi
mikrobiyoloji
Ekim 29, 2018
CİTROBACTER TÜRKÇE
CİTROBACTER
Citrobacter türleri düzdür, fakültatif anaerobik, Gram-negatif basillerdir ve Enterobacteriaceae ailesindeki peritriköz flagella ve koliform bakteriler aracılığıyla tipik olarak hareketlidir. . Bu cins 1932'de Werkman ve Gillen tarafından önerilmiştir. 1993'ten önce sadece üç tür, Citrobacter freundii, Citrobacter koseri (Citrobacter diversus) ve Citrobacter amalonaticus kabul edildi. C. freundii bu cinsin tür cinsidir ve sonraki iki tür başka isimler olarak adlandırılmıştır. C. koseri, 1993 yılında Uluslararası Sistematik Bakteriyoloji Komisyonunun Yargı Komisyonu tarafından C. diversus'un yerini almıştır. Aynı yıl, Brenner ve ark. DNA hibridizasyonu ile 11 genomospesitasyona Citrobacter sınıflandırılmış: C.freundii, C.koseri, C. amalonaticus, C. farmeri, C. youngae, C. braakii, C. werkmanii, C. sedlakii ve isimsiz genomospecies 9, 10 ve 11. Daha sonra, genomospecies 9, 10 ve 11 C. rodentium, C. gillenii ve C. murliniae olarak adlandırıldı.
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| Citrobacter freundii Citrobacter türleri yaygın olarak suda, toprakta, gıdada ve hayvanların ve insanların bağırsaklarında bulunur. Birçok Citrobacter enfeksiyonları nozokomiyal olarak edinilir; bununla birlikte, aynı zamanda topluluk edinilmiş olabilirler. Geniş bir sürveyans çalışması, Gram-negatif enfeksiyonun% 0.8'inin Citrobacter spp. Hastane ayarlarında Citrobacter spp. Enterobacteriaceae'nin % 3 - 6'sını oluşturabilir nozokomiyal enfeksiyona neden olur. Citrobacter enfeksiyonları olan hastalarda, bakteriler anneden veya yatay olarak taşıyıcılardan veya diğer hastane kaynaklarından dikey olarak iletilebilir. Enfeksiyon sporadik vakalar veya nozokomiyal salgınlar olarak ortaya çıkabilir. Dikey veya nozokomiyal bulaşma, bazı sporadik olgularda bakterilerin kökenini ve aile üyeleri veya diğer kişiler için taşıyıcılar gibi taşıyıcılardan bulaşmayı açıklayabilir. 
LABORATUVAR TANI
Citrobacter enfeksiyonu olan hastalar sadece kültür tarafından tanımlanabilir ve onaylanabilir. Citrobacter türleri çeşitli kültür ortamlarında büyüyebilir. Tüm türler , gaz üretimi ile Citrobacter ferment glikozu olarak tanımlanmıştır . Birkaç istisna dışında, organizmalar hareketlidir ve sitrat kullanırlar . Farklı türler biyokimyasal testlerle ayırt edilebilir . Antimikrobiyal duyarlılık testinin yorumu Enterobacteriaceae için kullanılan kriterleri takip eder .
Organizmalar, muhtemelen ağız boşluğunu, alt gastrointestinal sistemi veya solunum yolunu kolonize ederler. Daha sonra, bakteriyemi ve merkezi sinir sistemi (CNS) enfeksiyonu dahil olmak üzere çeşitli alanların enfeksiyonu ile sonuçlanabilir. Hastane dışı salgınlar büyük ölçüde hastane personeli tarafından gastrointestinal ve el taşımalarına bağlıdır.
CNS için C. koseri'nin özel eğilimi iyi anlaşılmamıştır. 32 kilodalton spesifik bir dış zar proteininin sinir dokularında bu organizmanın tropizmi ile ilişkili olduğu ve menenjit ve apseye neden olduğu ileri sürülmüştür.
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28 Ekim 2018 Pazar
mikrobiyoloji
Ekim 28, 2018
Citrobacter
CITROBACTER SPP.
Citrobacter species are straight, facultative anaerobic, Gram-negative bacilli and are typically motile by means of peritrichous flagellae and coliform bacteria in the Enterobacteriaceae family. This genus was proposed in 1932 by Werkman and Gillen. Before 1993, only three species, Citrobacter freundii, Citrobacter koseri (Citrobacter diversus), and Citrobacter amalonaticus, were recognized. C. freundii is the type species in this genus, and the later two species have been called other names. C. koseri was accepted to replace the name C. diversus by the Judicial Commission of the International Committee on Systematic Bacteriology in 1993. In the same year, Brenner et al. classified Citrobacter into 11 genomospecies by DNA hybridization: C.freundii, C.koseri, C. amalonaticus, C. farmeri, C. youngae, C. braakii, C. werkmanii, C. sedlakii, and unnamed genomospecies 9, 10 and 11. Later, genomospecies 9, 10 and 11 were named as C. rodentium, C. gillenii, and C. murliniae.
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| Citrobacter freundii |
Citrobacter species are commonly found in water, soil, food, and the intestinal tracts of animals and humans. Many Citrobacter infections are nosocomially acquired; however, they can also be community acquired. A large surveillance study demonstrated that 0.8% of Gram-negative infection was caused by Citrobacter spp. In the hospital settings, Citrobacter spp. might account for 3 – 6% of all Enterobacteriaceae causing nosocomial infection. In patient with Citrobacter infections, the bacteria can be transmitted vertically from mother or horizontally from carriers or other hospital sources. The infection may occur as sporadic cases or nosocomial outbreaks. Vertical or nosocomial transmission may account for the origin of bacteria in some sporadic cases, and transmission from carriers such as family members or other contacts accounts for others.
LABORATORY DIAGNOSIS
Patients with Citrobacter infection can be identified and confirmed only by culture. Citrobacter species can grow in various culture medium. All species identified as Citrobacter ferment glucose with production of gas. With few exceptions the organisms are motile and utilize citrate. Different species can be differentiated by biochemical tests. The interpretation of antimicrobial susceptibility testing follows the criteria used for Enterobacteriaceae.
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| GRAM STAINING |
The organisms probably colonize the oral cavity, lower gastrointestinal tract, or respiratory tract first. Later it may result into infection of various sites, including bacteremia and central nervous system (CNS) infection. Nosocomial outbreaks are largely due to gastrointestinal and hand carriage by hospital personneln.
The particular propensity of C. koseri for CNS is not well understood. A specific outer-membrane protein of 32-kilodalton was proposed to be related to the tropism of this organism for nervous tissues and causing meningitis and abscesses.
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| Citrobacter freundii on MacConkey |
27 Ekim 2018 Cumartesi
Yersinia Pestis
Ekim 27, 2018
YERSINIA SPP.
YERSINIA
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| GRAM STAINING |
YERSINIA PESTIS
Yersinia Pestis: Causative agent of plague.
General characterstics
Gram negative bacilli or coccobacilli, with rounded ends, convex or parallel sides. Non motile. Capsule present when grown at 37ºC. Bipolar staining (safety pin appearance) with methylene blue or giemsa stain.
Pleomorphic when grown in unfavourable conditions (nutrient agar with 3% sodium chloride). Grow on ordinary media. Colonies are dark brown on blood agar because of absorption of haemin. On Maconkey agar NLF colonies are produced. Optimum temperature 27ºC Liquid media- granular deposit and surface pellicle. Shows stalactite growth if a drop of sterile oil/gheebroth is allowed to float on broth and the medium is not disturbed growth hangs down from oil/ghee into the liduid medium which looks like stalactites.
Catalase: Positive
Oxidase, Urease, and Indole: Negative
- Fraction-1 or F1: heat labile protein envelope antigen ⇒ antiphagocytic
- V and W antigens: always produced together ⇒inhibits phagocytosis andintracellular killing,
- Pesticin 1 (bacteriocin), fibrinolysin ,coagulase : inhibits strains of Y.enterocolitica and Y. Pseudotuberculosis and E.coli
- Plague toxin (endotoxin -LPS and Murinetoxin)Ability to synthesize purine
Pathogenesis
Causative agent of plague. It is a zoonotic disease. Rodents are the natural reservoirs. It is transmitted through a bite of an infected rat flea (Xenopsylla species), but can also be transmitted by air (especially during pandemics of thE disease). Rat fleas become infected after taking blood meals from septicemic animals. Y. pestis grows in the midgut and eventually blocks the proventriculus, starving the flea for blood. The insects attempt to feed more often but end up giving back infected blood into the wound. Rat flea can’t fly. It jumps to a height of <2 feet, usually biting on the legs of humans.
Laboratory Diagnosis
Specimen
- For pneumonic plague Sputum /Bronchial wash/ tracheal aspirate,
- For septicemic plague - blood.
- For bubonic plague - Aspirate or biopsy of bubo.
Culture: Exudates/other specimen cultured on blood agar. Perform biochemical testing for identification of the isolate. Blood culture can be done in septicemic cases.
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| Yersinia enterocolitica growing on C.I.N. Agar |
YERSINIA ENTEROCOLITICA
Causative agent of gastroenteritis, mesenteric lymphadenitis and septicaemia. Laboratory diagnosis is by isolation of organism from blood, lymph node, feces, food or soil or by serology using tube agglutination test. Cold enrichment often helps. The organism is motile at 25°C but non motile at 37°C.
YERSINIA PSEUDOTUBERCULOSIS
Causative agent of pseudotuberculosis (a zoonosis). Causes mesenteric lymphadenitis and erythema nodosum especially in young males. Diagnosis is by serology using tube agglutination test (for antibody detection). This organism is motile at 25°C but non motile at 37°C and hydrolyses urea and has relatively poor growth on MaConkey agar.
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