기초 미생물학3

복습-Macromolecular interactions-공유결합Hydrogen bonds (H-bonds)  Ionic interactions (salt bridges)  van der Waals interactions  Hydrophobic interactions  Disulfide bonds
Molecules in water Most biological activities occur in aqueous (water-based) solutions • 친수성Hydrophilic molecules (contains O or N in many cases) -dissolve in water • 소수성Hydrophobic molecules (consist of only C and H) repel water •둘다가지고있음 Amphipathic molecules -have both hydrophilic and hydrophobic properties
Carbohydrate Bonds• Functions –cell structure, adhesion, and metabolism연결,부착,변형,
Lipids Functions – Triglycerides소수성 –energy storage – Phospholipid –major cell membrane-안정성 유지charge component – Steroids –cell membrane component-microplasma
Proteins-서로다른 amino acid Formation of a peptide bond=-Amino acids are attached through peptide bonds to form proteins.=peptide bond 선형상으로 연결하고,여러가지 결합력Primary structure Secondary structure Tertiary structure Quaternary structure
Primary structure Secondary structure Tertiary structure Quaternary structure-단위체-dnarna
Chapter 3 Tools of the Laboratory: The Methods for Studying Microorganisms=현미경 원리 종류 염색법Battling a brain infection- 50-year-old woman  Flu-like symptoms (fever, aching joints, sore throat, headache)  Overall normal according to an initial examination  Later out of consciousness, dark brown spots on her legs  Cloudy spinal fluid  Gram negative bacteria  Tiny pairs of red cocci(diplococci)  A blood culture (bacteria in her bloodstream)  White blood cells carrying same cocci inside  Cerebrospinal fluid (CSF) culture  Neisseria meningitidis독감-반점-척수액을 뽑음- 미생물-그람염색법-두개의 구가 뭉쳐있음-‘임질 뇌수막염’
What is responsible for this disease?- Gram stain of Bacillus anthracispositive 안쪽 산소요구도- 미생물이 어떻게 감염됐을지
Optical microscope (bright-field)-광학현미경-눈을통해 확인할 수 있다
Principles of Light Microscopy– The objective lens forms the magnified real image – The real image is projected to the ocular where it is magnified again to form the virtual image • 확대 Total magnificationof the final image is a product of the separate magnifying powers of the two lensespower of objective x power of ocular-접안렌즈*대물렌즈The Microscope Key characteristics of a reliable microscope are: • Magnification확대–ability to enlarge objects (extent of enlargement) • Resolving power (resolution)–ability to show detail해상도,디테일Resolution (resolving power)Low resolution High resolutionResolution(resolving power) defines the capacity to distinguish or separate two adjacent objects-훨씬더 자세하게 Resolution -wavelength불분명->두개의점을 구별 할 수 있는 최소의거리 -수치로표현
 Shorter wavelength and larger numerical aperture provide higher resolution.  Visible light wavelength: 400-750nmvisible liight  NA (numerical aperture, 개구수): 0.1 –1.25 /2f-로나눔=최소의 거리  Oil immersion objectives resolution is 0.2 μm  Magnification for optical microscope: 40X~ 2000X virus는 잘안보임 배율을 광학현미경에서 얻을 수 있는 최대
Effect of wavelength on resolution 긴파장,작은파장(더 쉽게 구별)
Resolution-더낮을수록 높아짐
• The numerical aperture (NA) of objective lens is a measure of the ability to gather light and resolve fine specimen detail at a fixed object distance. Numerical aperture ranges from 0.1 (low power lens) to 1.25 (oil immersion lens). -Oil 굴절율 빛이직진has the same optical qualities as glass. -Principle of immersion microscopy. Path of rays with immersion medium (yellow) (left half) and without (right half). Rays (black) coming from the object (red) at a certain angle and going through the coverslip (orange, as the slide at the bottom)
Vrius는 보기 힘듦Concept Check: B. Short wavelengths of light-짧은거리높아짐
In order to get the best resolution possible with a light microscope, you would want:
A. Long wavelengths of light B. Short wavelengths of light C. The wavelength doesn’t affect resolution D. Low numerical aperture of objective lens
Variations on the Optical Microscope • Bright-field (일반광학)살아있는부분–most widely used; specimen is darker than surrounding field; live and preserved stained specimens 빛투과 밝 안투과어
• Dark-field (암시야)–brightly illuminated specimens surrounded by dark field; live and unstained specimens 반대
• Phase-contrast(위상차)–transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures빛이투과 섬모가 확연하게Differential interference contrast (DIC) microscope-색깔이 두가지 확연하게
• Similar to the phase contrast but has more refinements -Two prisms –contrasting colors -Two beams of light => Vivid color, three-dimensionalFluorescence (형광) Microscope • Uses fluorescence to generate an image • Uses dyes that emit visible light when bombarded with shorter lights –fluorescence • Useful in diagnosing infections Fluorescent dyes are bound to specific antibodies (fluorescent antibody) 형광-작은 chemical,색깔이바뀌어서 나옴 빛energy가흡수되어 방출된다
mouse macrophages (red) Intracellular Francisella tularensis (green) antigen 을 specitic하게 에너지를 방출 섞어주어 얼마나 증식되었는가도 확인 할 수 있다 두가지의 antibody로 반응하여 두가지색을 얻음
고유으색이존재Scanning confocal (공초점) 초점이 맺히는 부위가 작아짐 microscopy - Uses a laser beam of light to scan various depths in the specimen and deliver a sharp image focusing on just a single plane. -Capture a highly focused view at any level from the surface to the middle of the cell. -Reconstruction of 3-D structures of cells from the obtained images. Electron Microscopy • Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds • Electron waves are 100,000 times shorter than the waves of visible light • Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength • Magnification between 5,000Xand 1,000,000X훨씬더 높게 확대된다 laserbeam 좁은범위에대해서 초점은 맞힐수있다. Electron Microscopy • Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds • Electron waves are 100,000 times shorter than the waves of visible light • Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength • Magnification between 5,000Xand 1,000,000X 전자현미경-훨씬더 높게 확대Comparison of microscopes electron microscope 전파를 증폭시킴 , 흡수, viruse particle의모양 3차원적모양
Transmission electron microscopes (TEM; 투과전자현미경) –transmit electrons through the specimen. Needs thin specimen. Darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts.-전자가 투과하고 평면Scanning electron microscopes (SEM; 주사전자현미경) –provide detailed three-dimensional view. SEMbombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.-metal로 코팅,입체
TEM-2차원적인이미지- 새깔을 입힘Concept CheckA. Brightfield B. Phase Contrast C. Scanning Electron D. Transmission Electron E. Fluorescence정답EWet mounts and hanging drop mounts-염색액없-살아있는미생물Fixed mountsare made by drying and heating a film of specimen.-불에넣어고착,고정단계Stains Dyes create contrast by imparting a color to cells or cell parts• Positive staining –Positive stain sticks to a cells and gives them color. • Negative staining–microbe repels dye, the dye stains the background, e.g. Nigrosin(blue-black)-염색법 반대에 들러붙어 염색• Basic dyes –cationic, positively charged chromophore • Acidic dyes –anionic, negatively charged chromophoreStaining reactions of dyes-투과되어 염색Staining• Simple stains–one dye is used; reveals shape, size, and arrangementSimple stains (단순염색한개) Differential stains (분별염색두가지) Structural stains (구조염색부분적 Differential stains–use a primary stain and a counterstain to distinguish cell types or parts (examples: Gram stain, acid-fast stain, and endospore stain)다른종류Acid-fast bacteria (pink) vs not-acid-fast-bacteria (blue) Used to detect Mycobacterium tuberculosis-결핵균을구별Gram staining1차 염료peptidoglycan의 두께가다름 매염제고착됨 탈색제쉽게 밖으로 빠저나감 대조 염색다른색깔로염색->비교Staining
• Structural stains –show certain cell parts not revealed by conventional methods: capsule and flagellar stains-일부구조물Sterilization-autoclave 내부의온도120 Asepsis병원성미생물Medium(pl. media)배지Culture증삭Pure culture순수배양Terms related to microbial culture Colony-군집 콜로니 하나의 cell로부터 존재The 6 I’s of Culturing Microbes-확인,미생물정보수집,점중,분리,증식,관찰Inoculation- introduction of a sample into a container of media to produce a culture of observable growth - implantation of a tiny microbial sample (inoculum) into or upon culture media to grow microbes.ex피-inoculation culture접종,클수있는조건에 넣어줌Culturing Microbes – Isolation-plate에넣음 콜로니형성Streak plate technique (획선평판법)백금Pour plate technique 부음(Loop dilution technique)Spread plate technique (도말평판법spread떨어짐Inspection-pure culturemixed culturecontaminants순수,두가지,오염물질한가지,두가지,원치않은미생물이 섞임
Macroscopic appearance (우리눈으로 알아차림e.g. colony morphology) -Microscopic 현미경appearance (e.g. cell morphology, staining characteristics) -Biochemical tests ezyme product: nutrient requirement, products, enzymes -Genetic characteristics : DNA sequencing -Immunological testing : Antibody항체 안티바디와 반응
Identification-determination of the type of microbes by the followings
5/6. Culturing Microbes