POSTLAB DISCUSSIONS IN MICROBIO1 LAB

Compiled by: Fortune Lapira Torrecampo, RMT

MICROSCOPE AND OTHER INSTRUMENTS

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RESOLUTION
Ability of the lens to distinguish fine detail of the specimen Determined by the wavelength of light from the illuminator. A wavelength is the distance between the peaks of two waves. As a general rule, shorter wavelengths produce higher resolutions of the image seen through the microscope

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PHASE CONTRAST MICROSCOPE

-Bends light that passes through the specimen so that it contrasts with the surrounding medium -Bending the light is called moving the light out of phase -Since the phase-contrast microscope compensates for the refractive properties of the specimen, you dont need to stain the specimen to enhance the contrast of the specimen with the field of view

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-This microscope is ideal for observing living microorganisms that are prepared in wet mounted slides so you can study a living microorganism.
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FLUORESCENCE MICROSCOPE
Fluorescent microscopy uses ultraviolet light to illuminate specimens.

Some organism fluoresce naturally, that is, give off light of a certain color when exposed to the light of different color. Organisms that dont fluoresce naturally can be stained with fluorochrome dyes. When these organisms are placed under a fluorescent microscope with an ultraviolet light, they appear very bright in front of a dark background.

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ELECTRON MICROSCOPE
-Developed in the 1930s, the electron microscope uses beams of electrons and magnetic lenses rather than light waves and optical lenses to view a specimen. -Very thin slices of the specimen are cut so that the internal structures can be viewed.
-Microscopic photographs called micrographs are taken of the specimen and viewed on a video screen.

-Specimens can be viewed up to 200,000 times normal vision. -living specimens cannot be viewed because the specimen must be sliced.
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DARK FIELD MICROSCOPE

If the regularly used condenser is replaced with what is known as a darkfield condenser, illuminated objects are seen against a dark background (or dark field), and the microscope has been converted into a darkfield microscope
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MICROBIAL CONTROL

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Number of organisms initially present

Killing effectiveness is gauged by decimal reduction time
D value Time required to kill 90% of population under specific Conditions Effect of prior washing

MICROBIAL CONTROL
Physical Methods
I. HEAT
- coagulates protein A. Moist Heat - water aids in the disruption of covalent bonds 1. Boiling - 1000C 15 30 minutes - kills vegetative forms but not spores and viruses -Mechanism of Action: Denaturation
2. Fractional Sterilization 2.a Tyndallization - steam for 30 minutes in 3 consecutive days 2.b Inspissation - 75 800C for 2 hrs. on 3 successive days -Mechanism of Action: Denaturation

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3. Autoclaving - steam under pressure -1210C at 15 psi for 15 30 minutes -Mechanism of Action: Denaturation

4. Pasteurization - sterilization for milk -620C for 30 minutes followed by rapid cooling (Flash Pasteurization) -Mechanism of Action: Denaturation 5. Inspissation -75 to 800C for 2 hours fro 3 consecutive days (with incubation in between); used to sterilize culture media with egg, serum, or sugars -Mechanism of Action: Denaturation
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Pressurized steam
Autoclave Achieves sterilization at 121C and 15psi in 15 minutes

Effective against endospores

Prions destroyed at 132C for 4.5 hours

B. Dry Heat - kills by oxidation a. Hot air oven - 160 1800C for 1 2 hrs - time depends on the penetration of heat on objects to be sterilized - for metals and glasswares -Mechanism of Action: Oxidation

b. Direct Flame - usually for inoculating loops and needles -Mechanism of Action: Burning contaminants to ashes C. Incineration -used for swabs, dressings, wipes, etc. -Mechanism of Action: Burning materials to ashes

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II. FILTRATION
- physical separation of microbes from liquid Example:asbestos, Millipore, nitrocellulose - Mechanism of Action: Physical removal of bacteria from suspending liquid or gas - Usually used for substances that could not tolerate heat ( injectable drugs, vitamins, amino acids)

TYPES OF FILTERS FOR LIQUIDS: A. Membrane filters made of nitrocellulose or cellulose acetate with uniform pore diameters of 0.45um or 0.22 um B. Depth fileters made of porcelain, glass or fibrous material (cotton, asbestos, and paper); microbes are retained by tortuous passageway AIR FILTERS high efficiency particulate air (HEPA) filter removes almost all microbes >0.3um in dm. Used for specialized hospital rooms and in laminar flow hoods 11/18/2011

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Filtration
Liquid filtration
Membrane filters allow liquids to flow through

Filtration of air
High efficiency particulate air (HEPA) filter removes nearly all microbes from air
Filter has 0.3m pores to trap organisms

III. OSMOTIC PRESSURE

-Mechanism of Action: by plasmolysis Example: immersion of meat in salt solution Fruits and vegetables in sugar solution

-results in loss of water from microbial cells -Used in food preservation IV. SONIC VIBRATION, TRITURATION, AGITATION
- mechanical methods that disintegrate bacteria Sonic vibration sound waves Trituration grinding Agitation shaking

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V. COLD - Mechanism of Action: Decreased chemial reactions and possible changes in proteins - used in the preservation of food, drug, and culture Refrigeration bacteriostatic effect Deep freezing- between -500C and -950C Lyophilization water removed by high vacuum at low temperature; for long tern preservation VI. DESSICATION - Mechanism of Action: Disruption of metabolism - Used in food preservation, bacteriostatic, - Removes water from microbes

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VII Radiation
Electromagnetic radiation
Energy released as waves (microwaves, radio, gamma, X rays, ultraviolet light etc) Shorter wavelength, higher frequency = more energy Radiation can be ionizing or non-ionizing

Ionizing radiation
Gamma radiation X-rays Electron accelerators

Causes direct damage to DNA and potentially to cell membrane

Causes indirect damage by producing reactive molecules such as superoxide and hydroxyl radicals Used to sterilize heat sensitive materials Medical equipment, surgical supplies, medications Some endospores can be resistant

Ultraviolet radiation
Non-ionizing (NI) radiation
Damages DNA (causes thymine dimers)

Used to destroy microbes in air, drinking water and on surfaces Limitation

Poor penetrating power (thin films or coverings can limit its effectiveness)

Chemicals
Chemicals can be used to disinfect and sterilize
Called germicidal chemicals

React with vital cell structures and components

Proteins DNA Cell membrane

 Sterilants Potency of chemicals

Formulations generally contain more than one antimicrobial agent Regulated by
FDA
Antiseptics

Destroy all microorganisms

High-level disinfectants
Destroy viruses and vegetative cells but not endospores

Intermediate-level disinfectants
Kill vegetative cells fungi, most viruses but not endospores

EPA
Disinfectants

Grouped according to potency

Low-level disinfectants
Kill fungi, vegetative bacteria and enveloped viruses No effect on mycobacteria, naked viruses or endospores

II. Chemical Methods

ANTISEPTIC a chemical substance that prevents growth of bacteria by either inhibiting or destroying microbes used on the surface of skin or mucous membranes

DISINFECTANT kills many but not all microbes aims to kill diseasecausing microbes but not spore formers used on inanimate objects
BACTERIOSTATIC agents that inhibit the growth of bacteria BACTERICIDE agents that kill bacteria

SANITIZER agents that reduce bacterial numbers to safe levels

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VARIABLES OF DISINFECTION 1.Concentration 2. Time 3. Temperature 4. pH

1 CT

where: N = no. of surviving bacteria C = concentration T = time

Increased concentration Increased Time

Decreased Survivors

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CHEMICAL AGENTS A. Disruption of Cell Membrane Alcohols - denatures protein - disorganizes the lipid structure in the membranes Example: 70% isopropyl alcohol - requires water for maximal activity

Detergents/ soaps - surfactants interact with the lipid in the cell membrane and with the surrounding water - increases the surface tension Example: Quaternary ammonium (Quats or Zephiran)
Phenols - original disinfectant of Lister - denatures protein Example: carbolic acid/ cresol (Lysol)
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B. Inhibition of Protein Synthesis 1. Chloramphenicol - from Streptomyces venezuelae - for Gram + and Gram - bacteriostatic Disadvantage:suppresses the bone marrow (anemia) 2. Erythromycin - freezes the ribosome 3. Tetracycline - broad spectrum 4. Streptomycin - from Streptomyces griseus - for MTB and N. gonorrhoeae 5. Gentamycin, Lincomycin, Clindamycin and Aminoglycosides

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C. Injury to Cell Membrane 1. Amphotericin B 2. Polymyxin B - from Bacillus polymyxa - for Gram Example: Brucella abortus, Klebsiella pneumoniae
3. Tyrocidin 4. Garamicidin S 5. Imidazole and Triazole

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D. Inhibition of Nucleic Acid Synthesis 1. rifampin 2. Quinolones 3. Sulfonamides 4. Trimethoprim

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DIRECT EXAMINATION OF UNSTAINED SPECIMENS

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SALINE MOUNT

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HANGING DROP PREPARATION

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MOTILITY
TRUE MOTILITY BROWNIAN MOVEMENT

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CYTOLOGY

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Bacterial Morphology
SHAPE
Cocci in Latin it means berry, spherical in shape Bacilli it means little stick or rod in Latin, rod shaped Spiral rod shaped with convolutions Vibrio curved rods that resemble a comma in serpentine S shape Spirilla spirals or corkscrew in shape Spirochetes spiral with an ability to wriggle or flex Pleomorphic with no definite shape

 ARRANGEMENT
1. Singles 2. Pairs diplococci, diplobacilli - cell division occurs in a single plane 3. Chains Streptococci, Streptobacilli - divides on 2 planes end to end 4. Clusters staphylococci (grape-like) - divides in 4 or more planes 5. 4s tetrads - divides in 2 planes 6. 8s sarcina - divides in 3 planes 7. Pallisade side-by-side division - picket fence in appearance

PROKARYOTES AND EUKARYOTES

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The Bacterial Cell and its Structures

I. Cell Wall
- maintains cell shape - determines if the bacteria is Gram + or Gram - protects against osmotic pressure change

a. Peptidoglycan / Murein or Mucopeptide - thicker in Gram + - susceptible to tears, saliva and mucus due to the presence of the lysozyme
Functions: 1. maintains integrity of the cell wall 2. target of some antibiotics Ex. Penicillin *Protoplast - seen in bacteria whose peptidoglycan layer is removed but are still viable, this makes them resistant to penicillin

b. Teichoic Acid

- protein attached to the peptidoglycan layer - found in Gram + only Functions:

Antigenic

c. Outer Membrane - found in Gram negative only - composed of lipopolysaccharide (LPS), lipoprotein and phospholipids ***Periplasmic space - space between the outer membrane and the plasma membrane

Other Properties: 1. Endotoxin (LPS) 2. Antigenic - capable of producing Antibodies Ex. Streptococcus pyogenes (ASTO) Salmonella typhi (Typhidot) 3. Contains Proteins - regulates the passage of materials from inside to outside

II. Cytoplasmic Membrane / Plasma Membrane

- similar to eukaryotes (phospholipids bilayer) - fluid mosaic model

Functions: 1. Active Transport - requires energy - movement is against concentration gradient

2. Energy generation - oxidative, phosphorylation 3. Synthesis for the precursors of the Cell Wall 4. Secretion of enzymes and toxins **Mesosomes - invaginations of the cell membrane

II. Cytoplasmic Membrane / Plasma Membrane (continued)

Functions:
shown in cell division binding site of DNA

III. Cytoplasm
- contains the Cytosol or the Amorphous Matrix to which nearly all other functions not conducted by the cell membrane occur - it is where interior structures are suspended
CONTENTS: 1. Ribosomes - for protein synthesis - it is significant because it is the target of some antibiotics 2. Granules - storage sites of nutrients - also important for staining purposes Ex. Corynebacterium - Babes-Earnst Mycobacterium - Muchs Yersinia - Bipolar bodies 3. Nucleoid - single circular molecule of DNA - the essence of a Prokaryote

III. Cytoplasm (continued)

4. Plasmids - extrachromosomal circular molecule of DNA - it can replicate by themselves independent from the chromosomes - it can be integrated into the DNA of another nucleoid 2 Types of Plasmids

1. Transmissible - transferred from one bacteria to another thru CONJUGATION by the pili 2. Non-transmissible Functions: 1. Antibiotic Resistance - this is the reason why resistance of bacteria to antibiotics are carried from one bacteria to another 2. Resistance to heavy metals 3. Resistance to UV light

III. Cytoplasm (continued)

5. Transposons - also known as Jumping Genes - these are short straight extrachromosomal molecule of DNA Functions: 1. Drug resistance 2. Mutations

Specialized Structures
1. Capsule - an excretory product that is polysaccharide in nature Importance: a. Virulence - limits phagocytosis - encapsulated bacteria are pathogenic

b. Identification - in the Neufeld-Quellung Reaction for Streptococcus pneumoniae c. Production of Vaccines - due to its high antigenic properties
d. Adherence or Attachment

2. Flagella

Specialized Structures (continued)

Arrangement a. Atrichous - no flagellum b. Monotrichous - 1 flagellum at one pole c. Lophotrichous - tuft of flagella at one pole d. Amphitrichous - tuft of flagella at both poles e. Peritrichous - flagellated all over

- contains the protein flagellin - requires energy Function: Locomotion observed thru the hanging drop slide or in biochemical tests Importance: a. Chemotaxis - attraction to chemical stimuli
b. Identification

Specialized Structures (continued)

3. Fimbriae or Pili - straight and short appendages - contains the protein pilin - seen in Gram bacteria Importance: a. Attachment b. Sex Pilus - for conjugation

Specialized Structures (continued)

4. Glycocalyx or Slime Layer - composed of loose polysaccharides Function:adherence or attachment Ex. Streptococcus mutans

5. Spores - formed in response to adverse conditions in the environment Function: Protection Processes of Sporulation 1. Axial Filament formation 2. Forespore Septum formation - infolding of the cell membrane to produce a double membrane 3. Engulfment of Forespore - results to the synthesis of 2 special layers forming the cell envelope 4. Cortex Synthesis 5. Coat Deposition 6. Maturation 7. Lysis of Mother Cell

Specialized Structures (continued)

SPECIMEN COLLECTION

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Microbiological examination
Specimens for microbiological examination must be appropriate eg, sputum rather than saliva. In general, specimens should be collected into, and transported in, a sterile container. Aspirated pus may be transported in a syringe, which must be capped immediately the needle has been removed and disposed of safely. Specimens should be delivered promptly to the laboratory. Although many specimens will tolerate a delay of several hours if refrigerated, cerebrospinal fluid must be transported to the laboratory immediately, without refrigeration. Similarly, for the detection of Neisseria gonorrhoeae and other fragile organisms, special arrangements may be needed: eg, express delivery, inoculation of plates at the time and place of collection, provision of special transport containers. Special requirements, for individual tests, are noted in the Test listing

Tube Guide
Tube content Determination Instructions
Invert slowly several times to ensure mixing

Shape

Heparin
Produce blue background in blood smeer

Plasma testing
some general chemistry (Glucose, urea,Cr)

EDTA
Inhibit ALK, CK Unsuitable for Ca &coagulation

Routine hematology , blood cont, Retc. CT. Sickle test, Glyco HB, HBelectro, ACTH,AS

Invert slowly several times to ensure mixing

Plain, No additive

Hormones, General chemistry Blood group, RH, Cross match,, Serology & Allergy, Fibrinogen, PT, PTT, TT, ATIII, coagulation Screen Ensure tube fills correctly to volume on label

Sodium Citrate1:9
Calating Ca,Inhibit aminotransferase. ALK & stimulate ACP

Sodium Citrate1:4

ESR

Ensure the presence of anticoagulant, Invert slowly several times to ensure mixing

Plane

Urine & Stool analysis,

Plane

Urine & Stool culture

Media for maintenance of bacteria

Blood

1- clean the area from which the blood is collected by iodine 2- withdraw for 8-10 ml of blood 3- insert the blood immediately in the vial and bring it to the lab quickly (2-3 ml for children and 5-7 ml for adult)

Type of urine specimens

1) Random specimens (drug abuse) 2) First-morning (microscopic examination, bHCG, 8-hours)

3) 24-hours specimen
Some analyses produce in different time though 24 hours of collection morning or noon like Creatinine, protein, Ca, phosphors and electrolyte (the sample must be refrigerated)

4) clean-catch specimen (MSU) for bacterial culture

5) catheter specimen 6) suprapubic specimen especially for infant 7) urine collected from children

collection bags with hypoallergenic skin adhesive

If the sample left at RT

normal bacteria will multiply producing contaminated sample if the organism urase producer, ammonia release will increase Ph resulting in destruction of cells and cast

the bacteria will break down any glucose RBC, WBC, Cast will lyze Protein conc will alter Bilirubin and Urobilinogen oxidized-not detected
Uric acid and urate deposited to for oxalate or phosphate crystal

Microbiological sample
specimen container instruction
Early morning, cough deeply for sputum, for children gastric wash, delay of specimen TB, Pn, HI

sputum Throat swab

stool

Clean, wide nick Sterile swab

Wide nick

Not contaminated with saliva, no antibiotic by 8-hours

Must be fresh sample within 2 hours

Rectal swab

Blood culture

For cholera alkaline peptone Bld water culture bottle Clean and sterile

Take the sample when tempt high, use iodine for sterilization, mix it and must reach the lab early, never refrigerated

semen

3-7 days of sexual abstinence, no condom, time of collection and deliver to the lab within 2 hour

Criteria for rejection of specimens

oMissing or inadequate identiification

oInsufficient volume oSpecimen collected in wrong collection tube

oContamination oInappropriate transport and storage oUnknown time delay

Oropharyngeal (Throat) Swab

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Transporting Specimens from Field to Lab

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Gram staining
- a principal stain - most clinically significant bacteria are detected except: a. intracellular bacteria b. bacteria that lacks cell wall c. bacteria with insufficient dimensions to be resolved by light (i.e. spirochetes) - provides preliminary diagnosis for initial treatment

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Gram staining
Principle - Gram (+) bacteria have thicker peptidoglycan layer (40) with numerous teichoic acid cross-linkages than that of G (-) (1or 2) - teichoic acid prevents decolorization - G(+) bacteria may loose CW integrity by: - antibiotic treatment - old cells - use of autolytic enzymes

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Gram staining
Rule:
1. All cocci are Gram (+) except: Neisseria Branhamella Veilonella 2. All bacilli are Gram(-) except: Bacillus Clostridium Corynebacterium Erysipelothrix Lactobacillus Listeria Mycobacterium

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Gram staining
Reagents Crystal Violet Grams Iodine 95% Alcohol Safranin - initial stain - mordant - decolorizer - counter satin

Gram (+) Gram (-)

- stains BLUE / VIOLET - stains RED / PINK

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Acid Fast Staining

- other commonly used stain for light microscopy - for Mycobacterium species Principle - designed for bacteria whose cell wall contains long chain fatty acids (mycolic acid) - mycolic acid render the cells resistant to decolorization - Acid fast organisms may be Gram (+)

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Acid Fast Staining

Reagents 1. Carbol Fuchsin- initial stain 2. Acid Alcohol -- decolorizer HCl + ethyl alcohol 3. Mehtylene Blue - counter stain or Malachite Green a. Hot Method Ziehl - Neelsen - uses steam as a mordant b. Cold Method Kinyoun Modification - uses phenol or tergitol as mordant Acid Fast Bacilli- stains RED or PINK Non-Acid Fast Bacilli- stains BLUE or GREEN

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