1. Laboratory Equipment

2. Microscope Centrifuges Refractometer Incubators Pipettes Chemistry & Hematology Analyzers

3. MICROSCOPE

4. Clinical Microscope Binocular Compound Light Microscope – Binocular – 2 eye pieces – Compound – uses a combination of lenses to generate an image Used in the clinic to evaluate blood, urine, semen, bodily fluids, feces, and other specimens *Ideally* 2 microscopes – One for parasitology – One for hematology & cytology

5. Parts of the Microscope Eye Pieces (ocular lenses) Nosepiece Objective Lenses Stage Slide holder Travel Knobs/Mechanical Stage Control Knobs Fine adjustment knob Course adjustment knob Condenser Diaphragm Light source

7. Online Resource Wisconson Online http://www.wisc-online.com How to use a microscope

8. Wisc-Online

9. Ocular Lens First Point of Magnification 10x power of magnification These can be focused for the individual These can be moved closer together or farther apart Some contain a small arrow to “point” things Some contain a reticle – a scale used to measure objects

10. Photo: Nikon Objective Lens

11. Second Point of Magnification 3 – 4 objectives are typical, they revolve around the nosepiece 4Xscanning red 10xlow poweryellow 40xhigh dryblue 100xoil immersionwhite/grey

12. Total Magnification Ocular lens power X Objective lens power = Total Magnification Photo: http://botit.botany.wisc.edu/botany_130/Microscope/Magnification.html

13. Through the looking glass… When viewing an object through the microscope, the object will appear upside down and reversed The right side is seen on the left The left side is seen on the right Movement of the mechanical stage is also reversed, when you move the stage to the left, the object appears to move to the right

14. The Stage! Where the slide is placed for viewing Has a small arm to hold the slide in place Travel Knobs – allow the slide to be moved in 4 directions on the stage Often the slide holder will have 2 small ruler like markings, you can use this to “mark” a spot on your slide to view again later

16. Adjustment Knobs These move the stage towards or away from the objective lenses Coarse Adjustment Knob – Brings the specimen into focus – Use this with your smaller power objectives Fine Adjustment Knob – Fine tunes & increases the details of the specimen – Can be used with any of the objectives

17. Condenser This sits just below the stage (substage) It has 2 lenses that focus light from the light source onto the specimen being viewed Light is focused by raising and lowering the condenser Typically best if the condenser is kept at its highest position Without this haloes & fuzzy rings can appear around the specimen

18. Diaphragm Typically an iris type Controls the amount of light that is directed up at the slide More light typically clarifies the specimen Lower light will increase the contrast Photo: http://www.thorlabs.com/thorproduct.cfm?partnumber=SM3D50

19. Light Source Found in the base Should be on a “dimmer” like switch – You should be able to control the intensity of the light

20. CENTRIFUGES

21. Used to separate substances of different densities that are in a solution Liquid portion = supernatant Solid portion = sediment (pellet) Centrifuge

22. Centrifuge Types Microhematocrit Centrifuge Clinical Centrifuge – Horizontal or Swing Arm – Angled (Fixed) Head Centrifuge

23. Photo Right: http://www.austincc.edu/mlt/phb/phb_equipment.html Photo below: http://dc344.4shared.com/doc/Mb55dBh_/preview.html

24. Horizontal Head (Swing Arm) Photo left: http://loudoun.nvcc.edu/vetonline/VET133/fecal%20centrifugal%20flotation.htm Photo right: http://www.vetlab.com/PowerspinMX.htm

25. Horizontal Head 2 Disadvantages – Excess speeds can cause heat buildup and damage delicate specimens – Some mixing of the supernatant and pellet may occur when the specimen cup falls back into the vertical position

26. Angled (Fixed) Head Centrifuge Photo right: http://labscientific.com/Lab-Equipment/Centrifuge/MyFuge-Mini-Centrifuge/ Photo left: http://www.labequip.com/fixedangle-centrifuge-head.html

27. Can rotate at higher speeds without excessive heat buildup when compared to the swing arm centrifuge Angled (Fixed) Head Centrifuge

28. Centrifuge On/Off switch Tachometer (speed setting) – Note – some do not have this and will only spin at one speed Braking Device – Used to rapidly stop it – Should ONLY be used in case of equipment malfunction MUST MUST MUST Counterbalance! Specimens should be spun – At a specific speed – For a specific time

29. Centrifuge If run too fast or too long: – May rupture cells and destroy the morphologic features If run too slow or for not enough time: – Will not completely separate the specimen – Will not concentrate the sediment

30. REFRACTOMETERS Photo: http://www.warren-wilson.edu/~physics/PhysPhotOfWeek/20050401LaserRefraction/

31. Refractometer Measures the refractive index of a solution Refraction is the bending of light rays as they pass from one medium into another medium with a different optical density The degree of refraction is related to the concentration of solid material in the medium – total solids

32. Refractometer These are calibrated to a zero reading with distilled water at a temperature of 60 - 100°F No solution can be more dilute or have a lower concentration of dissolved solids than distilled water The wide range of temperature ensures reliability (even when the refractometer is held in your warm hands)

33. Photo left: http://www.aimscope.com/refractometers.htm Photo right: http://encyclopedia.che.engin.umich.edu/Pages/ProcessParameters/Refractometers/Refractometers.html Refractometers

34. Refractometer Used in clinical practice to determine – The specific gravity of urine and other fluids – The protein concentration of plasma or other fluids Clinical refractometers have build in calibrated scales for these measurements

35. 1. Inspect and clean the prism cover glass and cover plate. 2. Place a drop of sample fluid on the prism cover glass. 3. Point the refractometer toward bright artificial light or sunlight. 4. Bring the light-dark boundary line into focus by turning the eyepiece. 5. Read and record the result with the appropriate scale (specific gravity, protein). 6. Clean the refractometer according to the manufacturer's recommendations.

36. Take your reading at the distinct light-dark interface

37. INCUBATOR Photo: http://diychickencoops.com/hatching-eggs-under-a-broody-hen

38. Incubators Clinical Laboratory incubators for microbiology – Should keep a constant 37°C – Should have a way to monitor this temperature – Proper humidity should be maintained Dish of water will do the trick Be sure it is changed regularly Be sure it is cleaned regularly to avoid contamination of your samples

39. Incubators Photo: Sirois, Hendrix and. Laboratory Procedures for Veterinary Technicians, 5th Edition. Figure 1-19 Mosby Elsevier Health Science, 2007.

40. PIPETTES Photo: http://www.alphalabs.co.uk/product-information/special-offers/pastette-wallchart-for-vets.aspx

41. Pipettes Used to transfer (and measure) liquids Many different types! Our focus will be on 2 types – Transfer pipettes – Graduated Pipettes Key – do not allow fluid to enter the pipetting device (the tip of the pipette should always point down)

42. Pipettes Transfer Very useful for a variety of tasks Used when you do NOT need to accurately measure the volume of liquid transferred Graduated Used when critical volume measurements are needed Single volume = only delivers the one set volume Multiple volume = has a “dial” or other graduations that will allow you to choose the volume to be delivered

43. Transfer pipettes

44. Graduated pipettes

45. CHEMISTRY ANALYZERS Photo: http://www.petmd.com/dog/general-health/evr_dg_reading_the_blood_chemistry_panel

46. Chemistry Analyzers Be familiar with yours! Most use the principles of photometry as the basis of their operating principles Some may also use electrochemical methods

47. Spectrophotometers are designed to measure the amount of light transmitted through a solution Colorimeter – uses a filter to select the wavelength Reflectometer – detects light reflected off a substance rather than transmitted light Chemistry Analyzers – Photometry

48. Basic Components: Chemistry Analyzers – Photometry in General In our analyzers there is a reagent that interacts with the sample & then the machine obtains the result. This is typically done using one of 2 methods: end point or kinetic

49. Chemistry Analyzers - Photometry End Point Method The reaction between the sample & reagent will reach a final stable (end) point The measurement is taken at this point Kinetic Method Used to measure serum enzymes (typically) The enzyme & reagent interact & will produce an end product. The enzyme is not lost or changed in this interaction The measurements are taken at specific points in time and measure the end product The amount of end product is used to calculate how much enzyme should have been present to make that much product

50. These machines have specific electrode that are allowed to interact with just one ion As ions move across an area separate by a membrane & the difference in voltage is measured by the electrode This difference corresponds to the number of ions present in the sample. Chemistry Analyzers – Electrochemical

51. HEMATOLOGY ANALYZERS Photo: http://www.howitworksdaily.com/q-and-a/why-is-blood-red/

52. Hematology Analyzers Know your machine! Three general categories of machines in use in the veterinary clinic: – Impedance Analyzers – Laser-Based Analyzers – Quantitative Buffy Coat Analysis

53. Current is passed between 2 electrodes Electrolyte fluid on either side conducts the current Hematology Analyzers Impedance Method

54. Cells are counted as they move across the aperture The cells are poor conductors of electricity, so they impede (block) the flow of current while they are in the aperture These changes in current are used to determine the blood cell concentration This system can tell the difference in the sizes of cells Hematology Analyzers Impedance Method

55. Laser beams are used to determine the size and density of the sample Different cells will scatter the laser light differently The degree and direction of scatter allows the machine to determine cell types Hematology Analyzers Laser-Based Method

56. Photo: http://www.idexx.ca/view/xhtml/en_ca/equine/in-house-diagnostics/in-house-analyzers/lasercyte- hematology.jsf?SSOTOKEN=0

57. Special microhematocrit tube The machine uses the measurements of tube volumes to provide an estimate of concentration Will only provide partial differential count – Granulocytes and then lymphocytes/monocytes Best used for screening because they provide an estimation rather then an actual count Hematology Analyzers Quantitative Buffy Coat Method

58. Hematology Analyzers: Quantitative Buffy Coat Method

59. Questions?