Neden bakterileri boyamalıyız?

Bacteria have nearly the same refractive index as water, therefore, when they are observed under a microscope they are opaque or nearly invisible to the naked eye. Different types of staining methods are used to make the cells and their internal structures more visible under the light microscope. .

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Bakteriler transparan, mikroskop altında görülmesi zor Boyama ile: Hücre büyüklüğü, şekli, hücrelerin yerleşimi, Gram boyanma özelliği, kapsül varlığı, endospor varlığı incelenebilir

Basit boyama Gram boyama Aside dirençli boyama Kapsül boyası Spor boyası

Stains: Increases the contrast by binding selectively to certain cell or certain parts of cells making them readily visible

• Bacteria are slightly negatively charged at pH 7.0 – Basic dye stains bacteria – Acidic dye stains

background

• Simple stain – Aqueous or alcohol

solution of single basic dye

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Bazik pozitif yüklü, bakteriler negatif yüklü

Asidik negatif yüklü Mordant hücrelerin boyaya olan

afinitesini arttırır

Bazik boyalar

Safranin

Bazik fuksin

Kristal viyole

Metilen mavisi

Asidik boyalar

Eozin

Asid fuksin

Kongo kırmızısı

Boyalar

• Pozitif ya da negatif yüklü organik tuzlardır

• Bir iyon renklidir: Kromofor (chromophore)

• Bazik boya: Pozitif iyon renklidir

Metilen Mavisi+ Cl-

• Asidik boya: Negatif iyon kromofordur

Kromofor grubu pozitif yüke sahiptir. Bu tür boyalara katyonik boyalar da denir. Örnek: Metilen mavisi bazik bir boyadır. İçeriği:tetramethhyl-thionin-chlorhydrate’tır. Tetramethyl-thionin baz’ı hidroklorik asit ile nötr bir tuz

meydana getirecek şekilde birleşmiştir. Tetramethyl-thionin bazı renklidir ve metilen mavisi boyasına

rengini verir, anyon kökü olan hidroklorik asit ise renksizdir Bakterilerin yüzeyi negatif olduğu için ayrıca DNA’da bulunan

fosfatların da negatif yüklü olması nedeniyle katyonik özelliğe sahip olan bazik boyalarla boyanırlar.

Bazik boyalar

Boyayıcı kısımları negatif elektriksel yüke sahiptir. Bu tür boyalara anyonik boyalar da denir. Asitik boyalar zemini boyamak için kullanılmaktadır. Asit fuksin, anilin blue, kongo red, çini mürekkebi, nigrosin, malaşit yeşili …

Asidik boyalar

1. Basit boyama bazik boya

2. Ayırt edici boyama M.o.ları birbirinden ayırt edilmesini sağlar

primer boya dekolorize zıt boya

3. Özel boyama yöntemleri

Leifson – flajel boyama

Negatif - kapsül boyama

1. Preparat hazırlanır: İncelenecek örnek temiz kuru bir lam üzerine yayılır. Yayma için öze, lam veya lamel kullanılır. Daha sonra lam havada kurutulur. 2. Fiksasyon yapılır: Amaç, lam üzerinde kurumuş olan bakterilerin lama yapışmasını sağlamaktır. Böylece boyama esnasında akıp gitmezler. fiziksel fiksasyon (tespit): yüzey üstte kalacak şekilde 2-3 kere bek alevden geçirilir. kimyasal fiksasyon (tespit): metil alkolde 3 dk bekletilir, kurutulur. 3. Boyama yapılır.

Boyama için;

Yaymanın hazırlanması

13 Dr.T.V.Rao MD

AMAÇ:

Hücrelerin şeklinin korunmasını sağlamak

Sometimes heat fixation is used to kill, adhere, and alter the specimen so it will accept stains

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Tespit edilmesi (fixation)

Isı ile tespit etme (Heat fixing) • Bakteriyi lama fikse eder

• Canlı olabilecek mo’ları öldürür

• Hücre membranı ve duvarının boyaya daha geçirgen hale gelmesini sağlar

Basit boyama

• simplest, the actual staining process may involve immersing the sample (before or after fixation and mounting) in dye solution, followed by rinsing and observation.

• The stain can be poured drop by drop on the slide

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• Methylene blue, Basic fuchsin

• Provide the color contrast but impart the same color to all the organisms in a smear

• Loffler's methylene blue: Sat. solution of M. blue in alcohol - 30mlKoH, 0.01% in water -100mlDissolve the dye in water, filter. For smear: stain for 3’.

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Basit boyama

Simple Staining Easier to Perform

But has Limitations

• Simple easy to use; single staining agent used; using basic and acid dyes.

• Features of dyes: give coloring of microorganisms; bind specifically to various cell structures

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Ayırt edici boyalar Differential Stains use two or more stains

and allow the cells to be categorized into various groups or types.

Both techniques allow the observation of cell morphology, or shape, but differential staining usually provides more information about the characteristics of the cell wall (Thickness).

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Gram staining

• Named after Hans Christian Gram, differentiates between Gram-positive purple and Gram-negative pink stains and is used to identify certain pathogens.

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Hans Christian Joachim Gram (September 13, 1853 - November 14, 1938) In Berlin, in 1884, he developed a method for distinguishing between two major classes of bacteria.

1880: Christian Gram

1880: Christian Gram

Gram – and + Cell Walls

Bakteri hücre yapısı: a)- Gram pozitif bakterilerin hücre çeperinde Gram negatiflere kıyasla oldukça kalın peptidoglikan tabaka mevcuttur. Bu nedenle aldıkları boyayı alkolle dekolarizasyonda gram negatiflere nazaran daha geç bırakırlar. b)- Gram pozitif bakterilerin hücre çeperinde karbonhidratlar, gram negatiflerde lipidler fazladır. Karbonhidratlar alkolle dekolarizasyon esnasında dehidratasyona (su molekülü açığa çıkar) uğrar. Porlar iyice büzüşür , daralır ve boya dışarı çıkamaz. Lipitler için ise alkol çözücüdür ve hücre çeperinde olan porlar daha çok açılacaktır. c)- Gram pozitif hücre çeperinde bulunan teikoik asit, bazik boyalarla daha kuvvetli birleşik oluşmasını sağlar.

Gram Staining Steps 1. Crystal violet :primary stain. Crystal violet may also be used as a simple

stain because it dyes the cell wall of any bacteria.

2. Gram’s iodine acts as a mordant (Helps to fix the primary dye to the cell wall).

A mordant is a substance used to set dyes on fabrics by forming an insoluble compound with the dye. Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl – ) ions. These ions penetrate through the cell wall and cell membrane of both Gram (+) and Gram(-) cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple. Iodine (I – or I3

– ) interacts with CV+ and forms large complexes of crystal violet and iodine (CV – I) within the inner and outer layers of the cell.

3. Decolorizer is used next to remove the primary stain (crystal violet) from Gram(-) bacteria (those with LPS imbedded in their cell walls). Decolorizer is composed of an organic solvent, such as, acetone or ethanol or a combination of both.)

4. A counter stain (Safranin), is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization

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Differential Stain: The Gram Stain

Gram Boyama Yönteminin Yararları 1- Mikroorganizmaları gram pozitif ve gram negatif diye iki ana gruba ayırır. 2- Mikroorganizmaların morfolojisi ve dizilimi esas alınarak ön tanı konur. Buna dayanarak nisbeten uygun bir antibiyotik seçilir, izolasyon için uygun besiyeri seçimi yapılır. 3- Örneğin usulüne uygun olup olmadığı hakkında bilgi verir.

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4

2

3

5

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Gram Stain

Gram pozitif

Gram negatif

Winn’s Law

• All gram-positive organisms aren’t blue

• All gram-negative organisms aren’t red

• All cocci aren’t round

• All bacilli aren’t long

• Bonus: Some bacteria stain poorly

• Super Bonus: Some bacteria don’t stain at all

Finally, a counterstain of basic fuchsin is applied to the smear to give decolorized gram-negative bacteria a pink color. Some laboratories use safranin as a counterstain instead. Basic fuchsin stains many Gram-negative bacteria more intensely than does safranin, making them easier to see. Some bacteria which are poorly stained by safranin, such as Haemophilus spp., Legionella spp., and some anaerobic bacteria, are readily stained by basic fuchsin, but not safranin.

Acid-Fast Stain

• Acid-fast cells contain a large amount of lipids and waxes in their cell walls

– primarily mycolic acid

• Acid fast bacteria are usually members of the genus Mycobacterium or Nocardia – Therefore, this stain is

important to identify Mycobacterium or Nocardia

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Aside dirençli hücre duvarı

Ziehl-Neelsen stain

• Ziehl-Neelsen staining is used to stain species of Mycobacterium tuberculosis that do not stain with the standard laboratory staining procedures like Gram staining.

• The stains used are the red colored Carbol fuchsin that stains the bacteria and a counter stain like Methylene blue or Malachite green.

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Ziehl- Neelsen Procedure 1. Make a smear. Air Dry. Heat Fix. 2. Flood smear with Carbol Fuchsin stain

– Carbol Fuchsin is a lipid soluble, phenolic compound, which is able to penetrate the cell wall

3. Cover flooded smear with filter paper 4. Steam for 10 minutes. Add more Carbol Fuchsin stain

as needed 5. Cool slide 6. Rinse with DI water 7. Flood slide with acid alcohol (leave 15 seconds). The

acid alcohol contains 3% HCl and 95% ethanol, or you can declorase with 20% H2 S04 – The waxy cell wall then prevents the stain from being

removed by the acid alcohol (decolorizer) once it has penetrated the cell wall. The acid alcohol decolorizer will remove the stain from all other cells.

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Ziehl- Neelsen 8. Tilt slide 45 degrees over the sink and add

acid alcohol drop wise (drop by drop) until the red color stops streaming from the smear

9. Rinse with DI water 10. Add Loeffler’s Methylene Blue stain

(counter stain). This stain adds blue color to non-acid fast cells!! Leave Loeffler’s Blue stain on smear for 1 minute

11. Rinse slide. Blot dry. 12. Use oil immersion objective to view.

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Ziehl-Neelsen stain

4 5 6

7

1 2 3

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Aside dirençli boyama

Acid Fast Stain

• Mycobacterium smegmatis (pink) acid fast retain the carbolfuchsin dye

• Micrococcus luteus (blue) not acid fast decolorized with acid-alcohol conterstained with methylen blue

http://www.bact.wisc.edu/Microtextbook/images/book_3/chapter_3/3-17.jpg

Acid Fast Stain Mycobacteria

Kapsül boyası

Flajel boyası

Tannik asit tuzları

Flajel boyası

Spor boyası

Malaşit yeşili

Endospore staining (Schaeffer-fulton Method)

• Spore: green

• Vegetative cell: red

http://www.arches.uga.edu/~howie/MVC-052endoS.JPG

http://homepages.wmich.edu/~rossbach/bios312/LabProcedures/endospore.jpg

• Preparation of a Bacterial Smear-Air dry • Application of Primary Dye (Malachite

Green) w/ concurrent application of heat • Allow slide to cool • Rinse w/ water • Application of Counterstain (Safranin)

Endospore staining (Schaeffer-fulton Method)

• Prepare a smear of the bacteria Bacillus megatatium (a spore-producing organism)

• Flood the smear with malachite green • Do not allow the stain to evaporate or

completely evaporate.

Endospore staining…

• Remove from heat and allow slides to cool • Once the slides are cool (important) rinse

with water • Flood the sample with safranin (30-60

seconds) • Rinse the slide blot dry observe under

microscopy. 10x, 40x, 100x (oil immersion).

http://www.tgw1916.net/video_pages/Spore.html

http://www.answers.com/topic/endospore

PRESUMPTIVE IDENTIFICATION OF STAPHYLOCOCCI

Gram positive cocci in clusters provide a presumptive identification of staphylococci. One cannot distinguish coagulase-positive from coagulase-negative staphylococci;the anaerobic staphylococci (peptococci) have a similar appearance. If well-defined clumps of cocci are present in a clinical specimen and are extracellular, the identification of these organisms as staphylococci is quite accurate.

In this Gram-stained smear of a positive blood culture bottle one can see gram-positivecocci in pairs, chains (arrows), and clusters (arrowhead). It is often possible to differentiate streptococci (chains) from staphylococci (clusters). When they are seen together,however, it is best to remain non-committal, reporting only gram-positive cocci. Bothgenera may be present, but it is particularly important not to direct attention away from the possible presence of staphylococci by reporting streptococci even if they are thepredominant form, as the staphylococci will not respond to penicillin therapy. Group B beta hemolytic streptococci were recovered in culture in this case.

Evaluation of the morphology of cocci on smears made from growth on agar media may be misleading. This smear of a Group B streptococcus taken from a blood agar plate contains gram-positive cocci in pairs and chains, but tetrads and clumps are also present. If the morphology is uncertain, the gram stain reactionand morphology from a four-hour broth culture should be evaluated.

DISTINCTIVE AND CLASSIC MORPHOLOGY OF PNEUMOCOCCUS (defined as lancet-shaped diplococci)

Some authorities describe a “chain” as more than four cocci in a row, by which definition two paris of pneumococci that haven’t separated do not qualify as a “chain.” Of course, there are always exceptions! In this field the number of cocci in some chains “squeaks” over the arbitrary limit of the definition. Notice that one of the cocci is more elongated (“a bigger lance”) than others (arrow).

FILAMENTOUS GRAM POSITIVE BACILLI

In addition, the filamentous gram positive bacilli, of which Actinomyces sp and Nocardia sp. are the most important members, may fragment and resemble gram-positive coccobacilli.

LARGE PLUMP GRAM-POSITIVE BACILLI

Large plump gram-positive bacilli suggest the identification of either Clostri-dium sp or Bacillus sp., although some lactobacilli may be rather broad. The clinical situation often helps to provide a presumptive diagnosis. The clostri-dial species that most characteristically has this morphology is the most important pathogen, Clostridium perfringens (shown here).

SPUTUM SMEAR FROM A PATIENT WITH A MENINGOCOCCAL PNEUMONIA