Microscopy, Staining, and Classification

1. Chapter 3 Microscopy, Staining, and Classification

2. A compound microscope uses a series of lenses for magnification. Light rays pass through specimen and into objective lens (one of a series of objective lenses). Has one or two ocular lenses Most have condenser lens to direct light through specimen Total magnification = magnification of objective lens X magnification of ocular lens. Microscopy: The Instruments

3. Microscopy General principles of microscopy Magnification Resolution Contrast

4. Light Refraction and Magnification

5. Limits of Resolution Resolution is the ability of the lenses to distinguish fine detail and structure. The human eye resolves to about 0.2mm.

6. Increasing Resolution at High Magnification To achieve this goal, a small objective lens must be used. Cell Staining and immersion oil is often used to increase contrast with the background and reduce light scattering.

7. Contrast Differences in intensity between two objects, or between an object and background Important in determining resolution Cell Staining changes the path of light to the objective and increases contrast.

8. Light Microscopy Bright-field microscopes Dark-field microscopes Best for observing pale objects Specimen appears light against dark background Increases contrast and enables observation of more details Phase contrast microscopes Used to examine living organisms or specimens that would be damaged or altered by attaching them to slides or by staining them.

9. Fluorescent Microscopes Direct UV light source at specimen; causes the specimen to radiate energy back as a longer, visible wavelength UV light increases resolution and contrast Some cells and molecules are naturally fluorescent, while others must be stained Used in immunofluorescence to identify pathogens and to locate and make visible a variety of proteins

10. Fluorescence Microscopy

11. Electron Microscopy Light microscopes cannot resolve structures closer than 200 nm because shortest wavelength of visible light is 400 nm electron microscopes have greater resolving power and greater magnification Magnify objects 10,000X to 100,000X Provide detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms Two types Transmission electron microscopes Scanning electron microscopes

12. Uses electrons instead of light. Transmission Electron Microscopy (TEM) Ultrathin sections of specimens (non-living specimen). Electrons pass through specimen Electron Microscopy

13. Scanning Electron Microscopy (SEM) An electron gun produces a beam of electrons that scans the surface of a whole specimen. Secondary electrons emitted from the specimen produce the image.

14. TEM Image

15. SEM Image

16. SEM Image

17. SEM Image

19. Staining Increases contrast and resolution by coloring specimens with stains/dyes.

20. Staining Procedure Smear of microorganisms (thin film) air dried to slide and then fixed to surface by heat or chemical fixation Microbiological stains are usually salts composed of cation and anion and one is colored (chromophore) Acidic dyes stain alkaline structures; basic dyes stain acidic structures and are used more commonly.

21. Preparation of Specimens for Light Microscopy A thin film of a solution of microbes on a slide is a smear.

22. Bacterial cells are negatively charged due to negatively charged proteins and nucleic acids within the cell Stains are used to illuminate cells consist of chromophore that may contain a positive or negative ion. in a basic dye, the chromophore is a cation (+). Crystal Violet, Methylene blue, Safarin in an acidic dye, the chromophore is an anion (-). Negrosin Preparing Smears for Staining

23. Staining Simple stains Differential stains Gram stain Acid-fast stain Endospore stain Special stains Negative (capsule) stain Flagellar stain Fluorescent stains

24. Use of a single basic (+) dye is called a simple stain. A mordant may be used to hold the stain or coat the specimen to enlarge it. Simple Stains

25. Simple Stains

26. Negative Staining an acidic (-) stain is utilized. results in only the background being stained. aids in observing the shape and size of a cell

27. Negative (Capsule) Stain

28. The Gram stain classifies bacteria into gram-positive and gram-negative. Gram-positive bacteria tend to retain stains when treated with alcohol. Gram-negative bacteria are susceptible to alcohol treatment and are stripped of stain. Differential Stains: Gram Stain

29. Gram Stain

30. Gram Stain

31. Gram Stain

32. Gram Stain

33. Cells that retain a basic stain (carbolfucsin) in the presence of acid-alcohol are called acid-fast. Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with methylene blue) to see them. Differential Stains: Acid-Fast Stain

34. Endospores are stained with the primary stain malachite green. The vegetative cells (actively growing cells) are counterstained with safranin (pink cells). Differential Stains: Endospore Stain

35. Flagellar Stain

36. Overview of Staining

37. Chapter 3: Case Study #1 – A Tonsilitis Outbreak An outbreak of an infectious disease sent more than 25 children from a local day care to the hospital. Five of the children were hospitalized with an upper respiratory tract infection. The initial onset of the clinical signs and symptoms of the disease included fever, sore throat, and headache. Later, signs included creamy yellow pus produced on the tonsils (exudative tonsillitis). Some also developed infections in deeper tissue, or a high fever and bright-red rash. Questions: 1. Is this a gram-positive or gram-negative bacterium? Explain your answer.   2. Briefly outline the process of a gram-stain.   3. Describe the arrangement of cells in the specimen.

38. Chapter 3: Case Study #2 – An Tuberculosis Outbreak in an HIV ward In early July 1999. a 34-year old HIV-infected man housed in a dormitory in the prison was taken to the prison hospital with a 2-week history of fatigue, fever, abdominal pain, and cough with blood. His chest radiograph was normal. The staff neglected to obtain a sputum specimen for culture and the prisoner was returned to his dormitory. In mid-August 1999, the man was evaluated at a community hospital. A sputum sample was obtained and an acid fast stained was performed. Later that year, 31 inmates and a medical student who examined the patient exhibited signs of this infection. Of the 31 inmates, all 31 resided in the same dormitory as the original infected patient.   Questions: 1. Describe the acid-fast stain method. 2. What molecule is detected using an acid-fast stain? 3. What color will acid-fast (+) cells be? What color will acid-fast (-) cells be? 4. What genus of bacteria is the cause of this disease outbreak?