Studying the Microbial World (microscopes) Supplemental instruction Designed by Pyeongsug Kim ©2010 Picture from Fall 2010 For Dr. Wright’s Bio 7/27 Class Updated: August 07, 2010

Microbes are too small to see…Microscope Picture from Light microscopes The development light microscopes  contributed to the study of microbiology Designed by Pyeongsug Kim, © Electron microscopes

Microscope *Useful of a microscope depends on it’s resolution. ___________ the number of times an image is enlarged. 10x, 40x, 60x, 100x Magnification __________ a measurement of clarity, the smallest distance between two points on a specimen that can still be distinguished. Resolution Designed by Pyeongsug Kim, ©2009

Light microscope - glass lens; 1,000x Electron microscope - electron lens; 100,000x - shorter than the waves of visible light. Atomic force microscope -an atomic scale; no preparation need.Microscope / Designed by Pyeongsug Kim, © Picture from

Light microscope -Light pass through a specimen. ; 1000x -Some need to stain bacteria.; some don’t  increase contrast b/w microorganisms and surroundings. -Bright-field (most common) Phase-constrast, Interference, Dark-field, Confocal, Fluorensence Picture from Designed by Pyeongsug Kim, ©2010

Which type of microscope are you using in Bio27 Lab. class? Bright-field microscope or compound microscope A compound light microscope is also called a bright field microscope. We usually call compound microscope. Designed by Pyeongsug Kim, © ) ______________ - Most common - Fixed specimen(killed) or moving organism (live) eg) E.Coli stained by Scarlet Euglena in pond water… Picture from Bright-field microscope Tardigrades (water bears) under a compound microscope -Youtube.com

Compound Microscope Eyepiece (oculars) Arm Stage Opening of Stage Fine-adjustment Coarse-adjustment Base Illuminator Iris diaphragm Iris diaphragm lever Stage clips High-power objective Low-power objective Nosepiece Body tube In the Dr. Hughey’s Bio3 class slide

2) _____________ - amplifies differences in refractive index to create contrast. - a darker appearance of the denser material.  increasing contrast. does not require staining to view the slide -does not require staining to view the slide. -Makes unstained cells more readily visible. -Best for observing intracellular structures Phase-contrast Designed by Pyeongsug Kim, © Paramecium bursaria on phase contrast microscope Youtube.com Can we see microbes alive? ____YES! Light microscope (Cont’d)

Can we see microbes alive? ____ Designed by Pyeongsug Kim, © ) ___________ -Light is directed toward the specimen at an angle. -can see unstained cells  Makes unstained cells more readily visible. -stand out as bright objects against a dark background. Dark field YES! Paramecium Darkfield.avi Youtube.com Light microscope (Cont’d)

Designed by Pyeongsug Kim, © Bright-field – most widely used, specimen is darker than surrounding field Dark-field – brightly illuminated specimens surrounded by dark field Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures Light microscope (Cont’d)

4) ___________ - Nomarski DIC(differential interfence contrast) - Two light beams 3D image!! - 3D image!! Interference Rotifer; Synchaeta sp via DIC microscopy Youtube.com Can we see microbes alive? ____ YES! Designed by Pyeongsug Kim, © Light microscope (Cont’d)

5) ___________ -Light onto the specimen (not pass through)  cells attached to soil particles or other opaque materials -UV light -Fluorescent dyes and Tags Uses dyes that emit visible light when bombarded with shorter uv rays. Can stain intracellular structures Can stain live cells Can distinguish living and dead cells Can be used in quantitative analysis Fluorescence Designed by Pyeongsug Kim, © Can we see microbes alive? ____ YES!

6) ___________ -Light onto the specimen (not pass through) 3D image!! -3D image!! -Can see inside intact cell in DETAIL.  a miniature CAT scan for cells. - Cell division -may use fluorescent dyes  to see cellular location or to determine molecule compound. - For thick structure eg) community of organism. Confocal scanning laser CAT scan for brain Light microscope (Cont’d) Designed by Pyeongsug Kim, © Can we see microbes alive? ____ YES!

_______________ - Can magnify images in excess of 100,000X - Fine details of cell structure - Should be in vacuum. - TEM (Transmission electron microscope), SEM (scanning electron microscope) Electron microscopesMicroscope Picture from Dr. Wright’s Bio27 PPThttp:// Designed by Pyeongsug Kim, © Electron microscope has better resolution than light one. Can we see microbes alive? Nope!!

Electron microscope (Cont’d) 1)_____________________ -To see inside cell. (organelles) - transmits electrons through the specimen -Thin sectioning Freeze fracturing, freeze etching -Vacuum required. -Artifacts are concern. 2) ______________________ -To see surface of the cell. -Vacuum NOT required. - a whole, metal-coated specimen with electrons 3D image!! - 3D image!! Very expensive~~~~ TEM (Transmission electron microscope) SEM (scanning electron microscope) Designed by Pyeongsug Kim, ©  Should kill the specimen!!

Avian flu virus by scanning microscopes Designed by Pyeongsug Kim, © Picture from electron microscope image of a virus (bacteriophage ) Virus can be seen through a electron microscope. Flu virus by electron microscopes

Designed by Pyeongsug Kim, © ER(Endoplasmic recticulum) and ribosome can be seen through a electron microscope.

Atomic force microscope Designed by Pyeongsug Kim, © First ever real time film of DNA- enzyme interaction Youtube.com -Atomic scale eg. Interaction between DNA and enzyme. -Atomic force microscope has much better resolution than electron microscope. Picture from