BACTE 1

Soap

Germicidal

*Listeria monocytogenes*

* Catalase (+)
* 3 C’s: Chicken, Coleslaw, Cheese

Chlamydia

* When delayed: 4’C
* Freezing: -20’C

BAP

* Phenotypic
* Gram (+) colonies: Dry, white, sometimes gray
* Gram (-) colonies: Gray and moist

*Mycobacterium gordonae*

Destroyed by chlorine

DNase test

Utilizes 1N HCl

LOA test

* For Gram (+)
* For nonfermentative
* For Enterobacteriaceae

Na hypochlorite

Inactivates HBV (10mins) and HIV (2mins)

Inoculating needles

* Nichrome = F(+) on oxidase test
* Not longer than 5cm

Wire loop

* 2mm diameter
* 0.001mL urine

50k CFU/mL

Significant for UTI

Pregnant

↑ C. albicans 

↑ Lactobacillus

Cotton swab

* Carrier state
* Lawn a culture
* Toxic to Neisseria 
* Good for virus

Charcoal

Removes the toxin inoculated by cotton

Phenotype

* Basis of identifying organisms
* Gram stain and colonies

Stool

Not Gram stained

PCR

Most definitive method of identification

Somatic antigen

Basis of serotyping

Anton van Leeuwenhoek

* Father of microbiology
* Microscopist
* 1st to describe bacteria

Robert Koch

Germ theory: relationship of organisms to human disease

Louis Pasteur

Father of Modern Microbiology

Ehrlich

1st to use dyes for stain

Bacteria

* Ave. size: 0.4-2μm
* Reproduction: Binary fission (two-fold increase)

Cell wall

* Peptidoglycan (murein)
* Protoplast: wall less G(+)
* Spheroplast: wall less G(-)

Gram (+)

* Thick peptidoglycan
* Teichoic acid

Gram (-)

* Thin peptidoglycan
* LPS (Lipid A – exotoxin)
* Somatic antigen

Plasma membrane

* Site for energy synthesis (ATP)
* Osmotic/permeability barrier

Nucleoid

* Chromosome: dsDNA
* Plasmid: Extrachromosomal DNA

Plasmid

Carries the antibiotic-resistance gene

Drug-resistance

Chromosome and plasmid-mediated

Metachromatic granules

Food reserves

Ribosomes

* Prokaryotic: 70S
* Eukaryotic: 80S

Common pili

Bacterial adherence

Sex pili

Gene transfer

ESBL

By Gram (-) bacteria

Endospores

* Calcium dipicolinate 
* *Bacillus, Clostridium*

Flagella

* Monotrichous: one only
* Amphitrichous: one at both ends
* Lophotrichous: tuft at one end
* Peritrichous: all around bacteria (most common)

*Lactobacillus*

Aerotolerant anaerobes

Autotrophs/Lithotrophs

Inorganic compound as source of carbon (CO2)

Heterotrophs/ Organotrophs

* Organic compound as source of carbone (Glucose)
* Pathogenic bacteria

Temperature requirements

* Psychrophilic: 0-20’C (ref) 
* Mesophilic: 20-40’C (pathogenic) 
* Thermophilic: 40-60’C

pH requirement

* Acidophilic: *Lactobacillus acidophilus* (Doderlein bacillus)
* Neutrophilic: pH 7.2-7.6 (optimal) – pathogenic
* Basophilic: *Vibrio* (Halophilic)

Moisture

Humidophilic

Salt concentration

* Halophilic
* *Enterococcus and V. parahaemolyticus*

Respiration (Aerobic)

* Glucose → CO2 + H2O 
* Kreb’s cycle
* Electron transport chain

Oxidation (Aerobic)

Glucose → Acid

Fermentation (Anaerobic)

* Glucose → Acid/Alcohol
* Embden-Meyerhoff pathway (glycolysis)

Lag phase

Adjustment

Log/Exponential phase

* ↑ in growth rate (cell division)
* Susceptible to antimicrobial agents

Stationary/plateau phase

* No net growth
* Death = Live cells
* Depletion of nutrients
* Accumulation of toxic wastes
* Sporulation

Death/Decline phase

↑ Death rate

Staining

Bacteria stain more by basic stains

Capsule stain

* India ink
* Borris method
* Nigrosin method

Not Gram stained

* *Chlamydia and Rickettsia* = intracellular 
* *Mycoplasma and Ureaplasma* = no cell wall
* Spirochetes

Gram Stain (Hucker’s modification)

* Crystal violet = 1min
* Gram’s iodine = 1min
* Acetone-alcohol or 95% ethanol = 30secs-1min
* Safranin O = 30 secs

Gram (+) becomes (-)

* Over-decolorization
* Old dying
* Acidic iodine
* Penicillin: omits iodine

Gram (-) becomes (+)

* Under-decolorization
* Thick smear

Acid Fast staining methods

Smear = 2 x 3cm

Pappenheim’s

*M. smegmatis* vs. *M. tuberculosis*

Baumgarten’s

*M. leprae* vs. *M. tuberculosis*

Fite Faraco

* *M. leprae*
* Counterstain: Hematoxylin

Acid fast organisms

* *Mycobacterium*
* *Nocardia* = Mod. AFS (1% H2SO4 as decolorizer) 
* *Cryptosporidium*
* *Legionella micdadei*
* *Rhodococcus equi*

Ziehl-Neelsen (Hot method)

* Best AFS
* **C-A-M**


1. **Carbolfuchsin** = 1’ stain


1. Start timing: Vapor (10mins)
2. Heat = Mordant
2. **3% Acid alcohol** = Decolorizer


1. HCl + 95% etOH
2. Until no more stain (Max: 3mins)
3. **Methylene blue** = counterstain


1. 30secs to 1min

Results:

AFO = **Red**

NAFO = **Blue**

Kinyoun (Cold method)

* Not used
* **C-A-M**


1. **Carbolfuchsin** = 1’ stain 


1. Phenol, Tergitol = Mordant
2. **3% Acid alcohol** = Decolorizer
3. **Malachite Green** = Counterstain

Results:

AFO = **Red**

NAFO = **Green**

Auramine-Rhodamine (Fluorochrome)

* Most sensitive


1. **Auramine-rhodamine** = 1’stain 
2. **0.5% Acid alcohol** = Decolorizer
3. **0.5% KMnO4** = Counterstain

Results:

AFO = **Yellow fluorescence**

NAFO = **No fluorescence**

AFB

Read 300 fields

Special stains

* Capsule = Negative stain
* Spore = Dorner, Wirtz, Conklin
* Metachromatic granules
* Albert’s
* Loeffler’s Alkaline Methylene Blue (LAMB)
* __Flagella = Leifson__
* Nucleic acid = __Feulgen__
* Polar bodies (ex: Y. pestis) = __Wayson__ 
* Rickettsia = __Gimenez__
* Spirochetes = Levaditi

Phase contrast microscope

For study of living unstained organisms

Electron microscope

* For viruses
* Light source: Electrons
* 100,000x magnification
* Stains:
* Negative stain
* PTA
* Heavy metals (Gold, Silver)

Transmission EM

DNA, RNA, chromosomes

Scanning EM

Surface structures (cell wall, capsule)

Inverted Microscope

For tissue culture

Interference microscope

Dual light source

Non staining method

String’s test (3% KOH)

Pure culture

* Streak plate = overlap method
* Pour plate = Water and milk bacteriology
* Selective medium
* Animal inoculation = for virus, *Chlamydia, Rickettsia*

Mixed culture

2 or more organisms

Stock culture

Stored at refrigeratior or freezer (long term)

Liquid

Broth

Semi-solid

0\.5-1% agarSolid

Solid

2-3% agar

Biphasic

* Both liquid and solid
* Ex. Castañeda = Brucella

General purpose media

* Nonfastidious organisms


1. **Sheep BAP** = Hemolysis
2. **Horse BAP** = Haemophilus -Heat-stable, provides X-factor
3. **Nutrient agar**

Enriched media

* Solid
* Fastidous organisms


1. CAP = Heat-labile, provides X & V factor

Enrichment media

* Liquid


1. Selenite F
2. Alkaline peptone water
3. Thioglycollate broth

Differential media

1. BAP = hemolysis
2. MAC
3. EMB
4. XLD
5. HEA

Selective media

* Inhibitory media


1. TCBS
2. SSA
3. TMA
4. CBAP

Inhibitory agents

* Antibiotics
* Dyes, bile salts = inhibit Gram (+)
* Alcohol (PEA) = inhibit Gram (-)

PEA

Gram (+) bacteria

Columbia CNA

Gram (+) bacteria

Gonococci Agar (GCA)

Gram (-) cocci

Gentamicin BAP

*S. pneumoniae*

Bacitracin CAP

*H. influenzae*

Cystine Tellurite Blood Agar

*C. diphtheriae*

Cystine Blood Glucose Agar

*F. tularensis*

Cystine Trypticase Agar

Confirm: *Neisseria*

Charcoal Cephalexin Blood agar

*B. pertussis*

Bordet-Gengou Agar (Potato Blood Glycerol Agar)

*B. pertussis*

BCYE

*L. pneumophila*

McCoy

*Cl. trachomatis*

TSB

*Brucella*