BACTERIAL STAINING basic methods : BACTERIAL STAINING basic methods Dr. T.V. Rao MD 1 Dr.T.V.Rao MD Microscopy helps to Measure and Observe the Bacteria : Microscopy helps to Measure and Observe the Bacteria Measurement
Microorganisms are very small
Use metric system
Metre (m) : standard unit
Micrometre (m) = 1 x10-6 m
Nanometre (nm) = 1 x10-9 m
Angstrom (Å) = 1 x10-10 m 2 Dr.T.V.Rao MD Why we should be Stain Bacteria : Why we should be Stain Bacteria 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.
. 3 Dr.T.V.Rao MD Staining helps in observation of Bacteria : Staining helps in observation of Bacteria Microscopes are of little use unless the specimens for viewing are prepared properly. Microorganisms must be fixed & stained to increase visibility, accentuate specific morphological features, and preserve them for future use. 4 Dr.T.V.Rao MD Stains and Staining : Stains and Staining Bacteria are slightly negatively charged at pH 7.0
Basic dye stains bacteria
Acidic dye stains background
Aqueous or alcohol solution of single basic dye 5 Dr.T.V.Rao MD What is a Stain : What is a Stain A stain is a substance that adheres to a cell, giving the cell color.
The presence of color gives the cells significant contrast so are much more visible.
Different stains have different affinities for different organisms, or different parts of organisms
They are used to differentiate different types of organisms or to view specific parts of organisms 6 Dr.T.V.Rao MD Staining Techniques : Staining Techniques Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes. 7 Dr.T.V.Rao MD Smearing out of the sample : Smearing out of the sample 8 Dr.T.V.Rao MD Fixation : Fixation Fixation–which may itself consist of several steps–aims to preserve the shape of the cells or tissue involved as much as possible. Sometimes heat fixation is used to kill, adhere, and alter the specimen so it will accept stains 9 Dr.T.V.Rao MD Simple staining : Simple staining 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 10 Dr.T.V.Rao MD Simple staining : Simple staining Methylene blue, Basic fuchsin
Provide the color contrast but impart the same color to all the organisms in a smear
Loffler's ethylene blue: Sat. solution of M. blue in alcohol - 30mlKoH, 0.01% in water -100mlDissolve the dye in water, filter. For smear: stain for 3’. For section: stain 11 Dr.T.V.Rao MD Simple staining (cont..) : Simple staining (cont..) Dilute Carbol fuchsin:- Made by diluting Z-N stain with 10- 15 times its volume of water- Stain for 20-25 seconds, wash with water
Use: To demonstrate the morphology of Vibrio cholera
Polychrome methylene blue:
Use: M’Fadyean’s reaction - B. anthracis 12 Dr.T.V.Rao MD Simple Stains : Simple Stains 13 Dr.T.V.Rao MD Bacterial arrangement : Bacterial arrangement Clusters (group).
No special arrangement. 14 Dr.T.V.Rao MD Simple Staining Easier to Perform But has Limitations : 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 15 Dr.T.V.Rao MD Differential Stains : Differential Stains 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). 16 Dr.T.V.Rao MD Gram staining : Gram staining Named after Hans Christian Gram, differentiates between Gram-positive purple and Gram-negative pink stains and is used to identify certain pathogens. 17 Dr.T.V.Rao MD Slide 18: 18 Dr.T.V.Rao MD Gram staining - Principles : Gram staining - Principles Gram staining is used to determine gram status to classify bacteria broadly. It is based on the composition of their cell wall. Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. Gram status is important in medicine; the presence or absence of a cell wall will change the bacterium's susceptibility to some antibiotics.
Gram-positive bacteria stain dark blue or violet. Their cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria 19 Dr.T.V.Rao MD Gram Staining Steps : Gram Staining Steps Crystal violet acts as the primary stain. Crystal violet may also be used as a simple stain because it dyes the cell wall of any bacteria.
Gram’s iodine acts as a mordant (Helps to fix the primary dye to the cell wall).
Decolorizer is used next to remove the primary stain (crystal violet) from Gram Negative 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.)
Finally, a counter stain (Safranin), is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization 20 Dr.T.V.Rao MD Stains differentiates different groups of Bacteria : Stains differentiates different groups of Bacteria To distinguish different kinds of bacteria into separate groups based on staining properties
Two types: Gram stain & Acid-fast stain. 21 Dr.T.V.Rao MD Differential Stains: Gram Stain : Differential Stains: Gram Stain 22 Dr.T.V.Rao MD Gram Staining technique : Gram Staining technique 23 Dr.T.V.Rao MD Gram Staining Procedure : Gram Staining Procedure 24 Dr.T.V.Rao MD Gram Staining technique : Gram Staining technique 25 Dr.T.V.Rao MD Structure and Reactivity to Gram Staining. : Structure and Reactivity to Gram Staining. 26 Dr.T.V.Rao MD Slide 27: 27 Dr.T.V.Rao MD Cell structure differentiates Gram positive from Gram Negative : Cell structure differentiates Gram positive from Gram Negative 28 Dr.T.V.Rao MD Gm+ve cocci & Gm-ve bacilli : Gm+ve cocci & Gm-ve bacilli 29 Dr.T.V.Rao MD Gram-positive : Gram-positive Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counter stain (safranin or fuchsine) and appearing red or pink. Gram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall. Gram-positive cell walls typically lack the outer membrane found in Gram-negative bacteria. 30 Dr.T.V.Rao MD GRAM-POSITIVE BACTERIA : GRAM-POSITIVE BACTERIA GRAM-POSITIVE BACTERIA are characterized by having as part of their cell wall structure peptidoglycan as well as polysaccharides and/or teichoic acids. The peptidoglycans which are sometimes also called murein are heteropolymers of glycan strands, which are cross-linked through short peptides. 31 Dr.T.V.Rao MD What are Gram Negative Bacteria : What are Gram Negative Bacteria Gram-negative bacteria are those bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counter stain (commonly safranin) is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color. The test itself is useful in classifying two distinct types of bacteria based on the structural differences of their cell walls. On the other hand, Gram-positive bacteria will retain the crystal violet dye when washed in a decolorizing solution. 32 Dr.T.V.Rao MD Gram negative bacteria : Gram negative bacteria On most Gram-stained preparations, Gram-negative organisms will appear red or pink because they are counterstained. Due to presence of higher lipid content, after alcohol-treatment, the porosity of the cell wall increases, hence the CVI complex (Crystal violet -Iodine) can pass through. Thus, the primary stain is not retained. 33 Dr.T.V.Rao MD Structural Details of Gram –ve Bacteria : Structural Details of Gram –ve Bacteria 34 Dr.T.V.Rao MD Gram Negative Bacteria : Gram Negative Bacteria Also, in contrast to most Gram-positive bacteria, Gram-negative bacteria have only a few layers of peptidoglycan and a secondary cell membrane made primarily of lipopolysaccharide 35 Dr.T.V.Rao MD Common Questions : Common Questions 1 Principles of Gram Staining and its uses.
2 Name common/pathogenic Gram positive and Gram negative Bacteria
3 Why some bacteria are Gram positive and others Gram negative.
4 Why some bacteria cannot be stained by Gram’s Method of staining.
5 What is the Use of Gram staining in your future clinical practice. Dr.T.V.Rao MD 36 Slide 37: ACID FAST STAINING Dr.T.V.Rao MD 37 Mycobacterium are Acid Fast Bacilli : Mycobacterium are Acid Fast Bacilli Mycobacterium are Gram-resistant (waxy cell walls), non-motile, pleomorphic rods, related to the Actinomyces. Most Mycobacteria are found in habitats such as water or soil. However, a few are intracellular pathogens of animals and humans. Mycobacterium tuberculosis, along with M. bovis, M. africanum, and M. microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex. Each member of the TB complex is pathogenic, but M. tuberculosis is pathogenic for humans while M. bovis is usually pathogenic for animals. 38 Dr.T.V.Rao MD Acid-Fast Stain : 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 39 Dr.T.V.Rao MD Ziehl-Neelsen stain : 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. 40 Dr.T.V.Rao MD Acid-Fast Organisms : Acid-Fast Organisms Primary stain binds cell wall mycolic acids
Intense decolorization does not release primary stain from the cell wall of AFB
Color of AFB-based on primary stain
Counterstain provides contrasting background 41 Dr.T.V.Rao MD AFB Staining Methods : AFB Staining Methods Zeihl Neelsen’s-hot stain
Modifications 42 Dr.T.V.Rao MD Ziehl- Neelsen Procedure : Ziehl- Neelsen Procedure 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. 43 Dr.T.V.Rao MD Ziehl- Neelsen Procedure (continued) : Ziehl- Neelsen Procedure (continued) 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. 44 Dr.T.V.Rao MD Ziehl-Neelsen stain : Ziehl-Neelsen stain 4 5 6
7 1 2 3 45 Dr.T.V.Rao MD How the Acid fast bacteria appear : How the Acid fast bacteria appear 46 Dr.T.V.Rao MD Fluorochrome AFB Microscopy : Fluorochrome AFB Microscopy More rapid and sensitive
Specificity : same with sufficient expérience
Equipment cost , bulbs, technical demands
for busy labs
External quality assessment should be done if this method is performed 47 Dr.T.V.Rao MD Fluorescence and Bright-field Microscopy : Fluorescence and Bright-field Microscopy 48 Dr.T.V.Rao MD Fluorochrome AFB Microscopy : Fluorochrome AFB Microscopy Primary fluorochrome AFB fluoresces
Auramine O Green
Auramine O-Rhodamine B Yellow/orange
Acridine Orange Yellow/orange
Note: Color of AFB may vary with filter system on microscope 49 Dr.T.V.Rao MD Staining of M.lepra : Staining of M.lepra M.lepra are less acid fast than M.tuberculosis group of organisms,
The concentration of H2So4 is reduced to 5 % for decolorising 50 Dr.T.V.Rao MD Slide 51: ALBERT’S STAINING FOR C.diptheria Dr.T.V.Rao MD 51 Diphtheria is Serious Disease When you suspect Diphtheria : Diphtheria is Serious Disease When you suspect Diphtheria In all cases of suspected cases of Diphtheria, stain one of the smears with Gram stain
If Gram stained smear shows morphology suggestive of C.diptheria, proceed to do Albert staining which demonstrates the presence or absence of metachromatic granules. 52 Dr.T.V.Rao MD Appearance of C.diptheria : Appearance of C.diptheria C.diptheria are thin Gram positive bacilli, straight or slightly curved and often enlarged (clubbing) at one or both ends and are arranged at acute angles giving shapes of Chinese letters or V shape which is characteristic of these organisms (Fig 1). Present in the body of the bacillus are numerous metachromatic granules which give the bacillus beaded or barred appearance. These granules are best demonstrated by Albert’s stain. 53 Dr.T.V.Rao MD Albert staining : Albert staining Albert stain I
Toluidine blue 0.15 gmMalachite green 0.20 gmGlacial acetic acid 1.0 mlAlcohol(95%) 2.0 mlDistilled water 100 ml Albert stain II
Iodine 2.0 gmPotassium iodide 3.0 gmDistilled water 300 ml 54 Dr.T.V.Rao MD Albert staining Procedure : Albert staining Procedure Cover the heat-fixed smear with Albert stain I. Let it stand for two minutes.
Wash with water.
Cover the smear with Albert stain II. Let it stand for two minutes.
Wash with water, blot dry and examine. 55 Dr.T.V.Rao MD How the C.diptheria appear : How the C.diptheria appear To demonstrate metachromatic granules in C.diphtheriae. These granules appear bluish black whereas the body of bacilli appear green or bluish green. 56 Dr.T.V.Rao MD Flagellar - staining : Flagellar - staining Flagella are usually invisible under light microscopy, but their identification and anatomy are important in determining some pathogens. Certain chemicals that bind to the flagella are used in the staining process. The flagella color may change or an increase in contrast should make them visible. 57 Dr.T.V.Rao MD Endospore staining : Endospore staining The cell walls of endospores are impermeable to most chemicals, and being in the genera Bacillus and Clostridium, cause diseases such as anthrax, teatanus and gangrene. The staining process involves both a primary stain and a counterstain. 58 Dr.T.V.Rao MD Capsule - Staining : Capsule - Staining A stain used to reveal negatively charged bacterial capsules. The encapsulated cells will have a halo appearance under the microscope. 59 Dr.T.V.Rao MD Negative staining : Negative staining India Ink, Nigrosin
Organisms are not stained, only the background is stained
Unstained organisms stand out in contrast
Use: To demonstrate the capsule of Cryptococcus neoformans, Streptococcus pneumoniae 60 Dr.T.V.Rao MD Slide 61: 61 Dr.T.V.Rao MD Nigrosin used for Negative Staining : Nigrosin used for Negative Staining The negative stain is particularly useful for determining cell size and arrangement. It can also be used to stain cells that are too delicate to be heat-fixed. 62 Dr.T.V.Rao MD Slide 63: Programme designed by Dr.T.V.Rao MD for “e” learning in Microbiology for Medical and Paramedical students in Developing world
firstname.lastname@example.org Dr.T.V.Rao MD 63