TITLE: Microscopy (Using the microscope)
The setting up of a microscope is a basic skill of microbiology yet it is rarely mastered. Only when it is done properly can the smaller end of the diversity of life be fully appreciated and its many uses in practical microbiology, from aiding identification to checking for contamination, be successfully accomplished. The amount of magnification of which a microscope is capable is an important feature but it is the resolving power that determines the amount of detail that can be seen.
All optical microscopes share the same basic components:
The eyepiece is a cylinder containing two or more lenses to bring the image to focus for the eye. The eyepiece is inserted into the top end of the body tube. Eyepieces are interchangeable and many different eyepieces can be inserted with different degrees of magnification. Typical magnification values for eyepieces include 5x, 10x and 2x. In some high-performance microscopes, the optical configuration of the objective lens and eyepiece are matched to give the best possible optical performance.
This occurs most commonly with apochromatic objectives.
1. ocular lens, or eyepiece
2. objective turret
3. objective lenses
4. rough adjustment knob
5. fine adjustment knob
6. object holder or stage
7. mirror
8. diaphragm
9.Condenser
The objective lens - a cylinder containing one or more lenses, typically made of glass, to collect light from the sample. At the lower end of the microscope tube, one or more objective lenses are screwed into a circular nose piece which may be rotated to select the required objective lens. Typical magnification values of objective lenses are 4x, 5x, 10x, 20x, 40x, 50x and 100x.
The stage is a platform below the objective which supports the specimen being viewed. There is a hole in the center of the stage through which light passes to illuminate the specimen. The stage usually has arms to hold the slides.
The illumination source - below the stage, light is provided and controlled in a variety of ways. At its simplest, daylight is redirected with a mirror. Most microscopes, however, have their own controllable light source that is focused through an optical device called a condenser, with diaphragms and filters available to manage the quality and intensity of the light.
The complete optical assembly is attached to a rigid arm which in turn is attached to a robust U shaped foot to provide the necessary stability. The arm is usually able to pivot on its joint with the foot to allow the viewing angle to be adjusted. Mounted on the arm are controls for focusing, typically a large knurled wheel to adjust rough focus, together with a smaller knurled wheel to control fine focus.
Some microscopes have objective lenses, called an oil immersion lens. To use this lens, a drop of immersion oil is placed on top of the cover slip, and the lens is very carefully lowered until the front objective element is immersed in the oil film. Such immersion lenses are designed so that the refractive index of the oil and of the cover slip is closely matched so that the light is transmitted from the specimen to the outer face of the objective lens with minimal refraction. An oil immersion lens usually has a magnification of 50 to 100×. The actual power or magnification of an optical microscope is the product of the powers of the ocular (eyepiece), usually about 10×, and the objective lens being used. Compound optical microscopes can produce a magnified image of a specimen up to 1000× and, at high magnifications, are used to study thin specimens as they have a very limited depth of field.
How a microscope works
The optical components of a modern microscope are very complex and for a microscope to work, the entire optical path has to be set up and controlled very accurately. Despite this, the basic optical principles of a microscope are quite simple. The objective lens is, at its simplest, a very high-power magnifying glass i.e. a lens with a very short focal length. This is brought very close to the examined specimen so that the light from the specimen comes to a focus about 160 mm inside the microscope tube. This creates an enlarged image of the subject.
This image is inverted and can be seen by removing the eyepiece and placing a piece of tracing paper over the end of the tube. By carefully focusing a brightly lit specimen, a highly enlarged image can be seen. It is this real image that is viewed with the eyepiece lens that provides further enlargement.
In most microscopes, the eyepiece is a compound lens, with one component lens near the front and one near the back of the eyepiece tube. This forms an air-separated couplet. In many designs, the virtual image comes to a focus between the two lenses of the eyepiece, the first lens bringing the real image to a focus and the second lens enabling the eye to focus on the virtual image.
Limitations of light microscopes
At high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by diffraction rings. These are called Airy disks. The limit of resolution (Resolving power of a microscope) is therefore taken as the ability to distinguish between two closely spaced Airy disks (or, in other words the ability of the microscope to reveal adjacent structural detail as distinct and separate). It is these impacts of diffraction that limit the ability to resolve fine details. The extent of and magnitude of the diffraction patterns are affected by both the wavelength of light (ë) and the refractive materials used to manufacture the objective lens and the numerical aperture (NA or AN) of the objective lens.
There is therefore a finite limit beyond which it is impossible to resolve separate points in the objective field. Assuming that optical defects in the whole optical set-up are negligible, resolution is indicated with d. nxsiná = numerical aperture (NA). Usually, a ë of 550 nm is assumed, corresponding to green light. With air as medium, the highest practical NA is 0.95, and with oil, up to 1.5. In practice the lowest value of d obtainable is around 0.2 micrometres or 200 nanometers.
Bacteria and yeast
Yeast can be seen in unstained wet mounts at magnifications ×100. Bacteria are much smaller and can be seen unstained at ×400 but only if the microscope is properly set up and all that is of interest is whether or not they are motile. A magnification of ×1,000 and the use of an oil immersion objective lens for observing stained preparations are necessary for seeing their characteristic shapes and arrangements. The information gained, along with descriptions of colonies, is the starting point for identification of genera and species, but further work involving physiology, biochemistry and molecular biology is then needed.
Moulds
Routine identification of moulds is based entirely on the appearance of colonies to the naked eye and of the mycelium and spores in microscopical preparations. Mould mycelium and spores can be observed in unstained wet mounts at magnifications of ×100 although direct observations of 'mouldy' material through the lid of a Petri dish or specimen jar at lower magnifications with the plate microscope are also informative (but keep the lid on!). Routine identification of moulds is based entirely on the appearance of colonies to the naked eye and of the mycelium and spores in microscopical preparations.
Hints
Adjust the iris diaphragm to achieve optimum balance between definition and glare. Do not control light intensity by moving the sub-stage condenser, the position of which should be to focus the light on the specimen. Re-adjust the iris diaphragm for each objective lens. For looking at wet mounts of living specimens of protozoa, algae, moulds and even yeasts, the low power objective lens (×10) is often adequate but also necessary for locating and centering on an area of interest before turning to the high power objective lens (×40). Without altering the focus, turn to the high power lens and then finely re-focus. Use the oil immersion objective lens for examining stained preparations of bacteria.
Put one drop of immersion oil onto the preparation; a coverslip is not required. Remove the slide and wipe the oil immersion lens clean at the end of the practical session.
Protozoa and algae
Protozoa and algae are large organisms and therefore are readily visible at a magnification of ×10 to ×100 in unstained wet mounts. A magnification of ×100 is advantageous for observing natural samples that contain a variety of organisms, particularly as many are very motile. Identification of algae and protozoa is based entirely on their microscopical appearance. The common algae are green and non-motile; diatoms have a brown, sculptured outer layer of silica and move slowly. Protozoa are colourless and most are motile. Hayinfusions and cloudy vase water are rich in algae and protozoa, but clear samples of water are rarely rewarding.
LECTURE 2
TITLE: Preparation of Films / Smears of Microorganism
This practical exercise will be carried out in sequence. Firstly, the slide required for making the films and smears will be meticulously cleaned and dried.
Cleaning of Slides
You are supplied with glass slides that have been soaked in chromic acid solution for about 2 hours, and washing solution (Detergent). Wash the slides very well and check whether your slides is greasefree by attempting to spread a drop of water over its surface in a well thin film.
If a thin film cannot be made with a drop of clean water on the slide, then the slide is not grease free and should therefore be re-cleaned. Store the slides in methylated spirit or alcohol (ethanol) until it is required.
SMEAR PREPARATION AND SIMPLE STAINING
Introduction
Success at bacterial staining depends first of all on the preparation of a suitable smear of the
organisms. The first step in preparing a bacteriological smear differs according to the source of the organisms. If the bacteria are growing in a liquid medium (broths, milk, saliva, urine, etc.), one starts by placing one or two loopfuls of the liquid medium directly on the slide. From solid media such as nutrient agar, blood agar, or some part of the body, placing one or two loopfuls of water on the slide and then make use of a straight inoculating wire to disperse the organisms in the water. Bacteria growing on solid media tend to cling to each other and must be dispersed sufficiently by dilution in water; unless this is done, the smear will be too thick. The most difficult concept for students to understand about making slides from solid media is that it takes only a very small amount of material to make a good smear. When your lecturer demonstrates these steps, pay very careful attention to the amount of material that is placed on the slide.
Procedure:
One way of observing the details of bacteria including its morphology and size involves smear
preparation and simple staining. A bacterial smear is a dried preparation of bacterial cells on a glass slide. In a bacterial smear that has been properly processed.
(1) The bacteria are evenly spread out on the slide in such a concentration that they are adequately separated from one another
(2) The bacteria are not washed off the slide during staining.
(3) Bacterial form is not distorted. In making a smear, bacteria from either a broth culture or an agar slant or plate may be used.
If a slant or plate is used, a small amount of bacterial growth is transferred to a drop of water on a glass slide and mixed. The mixture, is then spread out evenly over a large area on the slide. One of the most common errors in smear preparation from agar cultures is the use of too large an inoculum. This invariably, results in the occurrence of large aggregates of bacteria piled on top of one another. If the medium is liquid, place one or two loops of the medium directly on the slide; and spread the bacteria over a large area. Allow the slide to air dry at room temperature. After the smear is dry, the next step is to attach the bacteria to the slide by heatfixing. This is accomplished by gentle heating, passing the slide several times through the hot portion of the flame of a Bunsen burner. Most bacteria can be fixed to the slide and killed in this way without serious distortion of cell structure.
The use of a single stain or dye to create contrast between the bacteria and the background is referred to as simple staining. Its chief value lies in its simplicity and ease of use. Simple staining is often employed when information about cell shape, size, and arrangement is desired. In this procedure, you are going to place the heatfixed slide on a staining rack, cover the smear with a small amount of the desired stain for the proper amount of time, wash the stain off with water for a few seconds, and, finally, blot it dry. Basic dyes such as crystalviolet (20 to 30 seconds staining time), carbolfuchsin (5 to 10 seconds staining time), or methylene blue (1 minute staining time) are often used. Once bacteria have been properly stained, it is usually an easy matter to discern their overall shape. Bacterial morphology is usually uncomplicated and limited to one of a few variations.
PREPARATION OF BACTERIAL SMEAR FROM AGAR SLANTS
Materials
24 to 48 hour tryptic soy broth or agar slants of Bacillus subtilis , and Staphylococcus aureus, microscope, clean microscope slides,• inoculating loop and needle, sterile distilled water, Bunsen burner, methylene blue, crystal violet (1% aqueous solution), Ziehl's carbolfuchsin ,wax pencil,immersion oil, slide holder
METHOD
Smear Preparation
1. With the wax pencil, mark the name of the bacterial culture in the far left corner on each of three slides.
2. For the broth culture, shake the culture tube and, with an inoculating loop, aseptically transfer 1 to 2 loopfuls of bacteria to the center of the slide. Spread this out to about an inch area. When preparing a smear from a slant or plate, place a loopful of water in the center of the slide. With the inoculating needle, aseptically pick up a very small amount of culture and mix into the drop of water. Spread this out as above. (Two slides should be prepared; one each of B. subtilis
Staphylococcus aureus.)
3. Allow the slide to air dry.
4. Pass the slides through a Bunsen burner flame three times to heat fix and kill the bacteria.
Simple Staining
1. Place the three fixed smears on a staining loop or rack over a sink or other suitable receptacle
2. Stain one slide with alkaline methylene blue for 1 to 1d minutes; one slide with carbolfuchsin for 5 to 10 seconds; and one slide with crystal violet for 20 to 30 seconds.
3. Wash stain off slide with water for a few seconds
4. Blot slide dry with blotting paper be careful not to rub the smear when drying the slide because this will remove the stained bacteria.
5. Examine under the oil immersion lens and report your observation
6. You may want to treat smears of the same bacterium with all three stains in order to compare them more directly. It is also instructive to cover bacterial smears for varying lengths of time with a given stain in order to get a feel for how reactive they are and the results of overstaining or understaining a slide preparation.
Preparing film from solidify medium
(a) With the solid material (i.e. the culture medium in a Petri dish, place a loopful or a drop of clean water on a sterile grease-free glass slide.
(b) Sterilize the wire loop and take a minute quantity of a distinct (discrete) colony of the culture by just touching the growth, and transfer to the drop of water on the slide.
(c). Emulsify thoroughly and spread the mixture evenly and thinly on the slide
(d). Dry and heat fix as described above. The film is ready for staining
TITLE: Preparation of Microbiological Stain
SIMPLE BACTERIAL STAIN E.G METHYLENE BLUE
Preparation of methylene blue
1. Dehydrated methylene blue
2. Distilled water
3. Measuring cylinder
4. Potassium hydroxide
5. Ethanol
6. Reagent bottle
7. Weighing balance
Purpose:-It is used to enhance the morphology of young bacterial cultures.
Procedure
Weigh 1g methylene blue and dissolve in 200ml of distilled water heated to 500c.
Add 2ml of 1% potassium hydroxide (KOH).
Add 60ml of ethanol, mix and filter using filter paper if there are suspended particles.
Store in a washed and dried reagent bottle
Label approximately and indicate the date of preparation.
GRAMS IODINE (MORDANT)
Weigh 1g of iodine, 2g of potassium iodine and 25ml of distilled water.
Dissolve the weighed iodine & potassium iodine in the distilled water, when the solids are fully dissolved, add more distilled water to make 300ml
Materials: Iodine, potassium-iodine, water, measuring cylinder, weighing balance, reagent bottle and label.
CRYSTAL VIOLET
Weigh 0.5g of crystal violet and dissolve in 100ml of distilled water. Pour into a cleaned and dried reagent bottles & label.
SAFRANIN
Preparation:
Weigh 10g of safranin crystals & dissolve in 5% ethanol
Uses: it is used as a counter stain in differential staining
Gram's staining. (For Hucker's modification of gram's staining). The most common and useful staining procedure used in bacteriological work is that of Gram.
Principle
All the modifications are based on the fact that certain groups of bacteria treated with one of the
basic para-rosaline dyes such as crystal violet, followed by addition iodine as the mordant, bleached with Alcohol/ Acetone) always retain the stain firmly; this means subsequent addition of secondary stain has no effect.
Such bacterial are termed gram positive while those bacteria which do not retain the stain are gram negative. The latter set of bacteria which is decolorised by alcohol/acetone may be interstained with a stain of contrasting colour, and more often a red stain such as carbol fuchsin or safranin are used.
Instruction
Taking appropriate precautions
Prepare 100ml of each of the Hucker's modification of Gram's staining solution
Crystal violet/ammonium oxalate solution serves as primary stain.
Solution A Solution B
2 gram crystal Violet (certified) 0.8 gram ammonium oxalate 20ml ethyl Alcohol
(95%) 80 ml distilled water
Mix solution A and B stored for 24hours filtered and kept at room temperature. A dark bottle is used.
Only certified crystal violet, gentian violet and methyl violet are not recommended as replacement for crystal violet because they contain impurities.
Iodine Solution (mordant)
Iodine Crystals 1g
Potassium Iodide 2g
Distilled Water 300ml
Grind Gram Iodine and Potassium Iodide in a mortal separately. Dissolve Potassium Iodide in a
flask, add small amount of water as possible. Add Iodine Crystal to Potassium Solution. When
dissolution is complete, add the remaining Distilled Water. Mix and allow to stand at room
temperature for 24 hours. Filter and store in a dark bottle away from direct sunlight.
Decolorizer
Acetone 1 volume
Ethyl alcohol (95%) I volume
Mix one (1) volume of acetone with one (1) volume of ethyl Alcohol and store in a sealed bottle.
Safranin (counter stain)
Safranin – 0.25g
Distilled water 90ml
Dissolve dye in ethyl alcohol, and then add distilled water to dye solution and all to stand at room temperature for 24hours. Filter and store away from direct sunlight
State the precaution taken during the preparations to get desired quality of stain.
TITLE: Gram Staining Techniques 1
LITERATURE REVIEW: In 1884 the Danish bacteriologist Christian Gram developed a staining technique that separates bacteria into two groups: those that are gram positive and those that are gram negative. The procedure is based on the ability of microorganisms to retain the purple color of crystal violet during decolorization with alcohol. Gram negative bacteria are decolorized by the alcohol, losing the purple color of crystal violet. Gram positive bacteria are not decolorized and remain purple. After decolorization, safranin, a red counterstain, is used to impart a pink color to the decolorized gram negative organisms. Although several explanations have been given for the Gramstain reaction results, it seems likely that the difference between gram positive and gram negative bacteria is due to the physical nature of their cell walls.
If the cell wall is removed from grampositive bacteria, they become gram negative. The peptidoglycan itself is not stained; instead it seems to act as a permeability barrier preventing loss of crystal violet. During the procedure the bacteria are first stained with crystal violet and next treated with iodine to promote dye retention. When gram positive bacteria then are decolorized with ethanol, the alcohol is thought to shrink the pores of the thick peptidoglycan. Thus the dye iodine complex is retained during the short decolorization step and the bacteria remain purple. In contrast, gram negative peptidoglycan is very thin, not as highly cross linked, and has larger pores. Alcohol treatment also may extract enough lipid from the gramnegative wall to increase its porosity further.
For these reasons, alcohol more readily removes the purple crystal violet iodine complex from gram negative bacteria.
Note: Crystal violet, the primary stain, causes both gram positive and gram negative organisms tobecome purple after 20 seconds of staining. When Gram's iodine, the mordant, is applied to the cells for one minute, the color of gram positive and gram negative bacteria remains the same: purple. The function of the mordant here is to combine with crystal violet to form a relatively insoluble compound in the gram positive bacteria. When the decolorizing agent, 95% ethanol, is added to the cells for 10–20 seconds, the gram negative bacteria are bleached colorless, but the gram positive bacteria remain purple. In the final step a counterstain, safranin, adds a pink color to the decolorized gram negative bacteria without affecting the color of the purple gram positive bacteria.
Of all the staining techniques you will use in the identification of unknown bacteria, Gram staining is, undoubtedly, the most important tool you will use. Although this technique seems quite simple, performing it with a high degree of reliability is a goal that requires some practice and experience.
Here are two suggestions that can be helpful: first, don't make your smears too thick, and second, pay particular attention to the comments that pertain to decolorization. Simple staining depends on the fact that bacteria differ chemically from their surroundings and thus can be stained to contrast with their environment. Bacteria also differ from one another chemically and physically and may react differently to a given staining procedure.
This is the principle of differential staining. Differential staining can distinguish between types of bacteria. The Gram stain does not always yield clear results. Species will differ from one another in regard to the ease with which the crystal violet-iodine complex is removed by ethanol. Grampositive cultures may often turn gram negative if they get too old. Thus, it is always best to Gramstain young, vigorous cultures rather than older ones. Furthermore, some bacterial species are gram variable. That is, some cells in the same culture will be gram positive and some, gram negative. Therefore, one should always be certain to run Gram stains on several cultures under carefully controlled conditions in order to make certain that a given bacterial “strain” is truly gram positive or gram negative. Indistinct Gram stain results can be confirmed by a simple test using KOH. Place a drop of 10% KOH on a clean glass slide and mix with a loopful of bacterial paste.
Wait for 30 seconds, and then pull the loop slowly through the suspension and up and away from the
slide. A gram negative organism will produce a mucoid string; a gram positive organism remains
fluid. As discussed earlier, gram staining techniques divide bacterial into two major group- Gram
positive and Gram negative.
Question
1. why is Gentian violet not used in any of the practical 1 and II as primary stain?
2. State the function of the other stains used
3. What is the function of acetone in the preparation?
Blog by SHERIF McCARLTON AlAODEEN TEMIYEMI (HOC TEMMY)
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