Dis

tingu

ishi

ng S

trai

nsIndividual bacterial species and strains may

be distinguished by:

• RFLP or rep-PCR analysis

• Protein profiling

• Immunological tests

• Phage typing

• Biochemical tests

This allows ID for treatment as well as for tracing disease outbreaks

For example, if it can be shown that two pathogens are identical (clonal) then it may be inferred that they came from the same place/person

RF

LP &

Rep

-PC

R

Pro

tein

Pro

file

Imm

unol

ogic

al T

ests • Slide agglutination antiserum and

slides (will be doing in lab)

• Enzyme-Linked Immunosorbent Assay (ELISA) antibodies and microplates (will discuss in detail last lecture)

• Western Blotting: antigens are separated by a gel-electrophoresis and detected using radioactive or fluoresence tagged antibodies

• Along with ELISA serves as basis for assay for HIV seroconversion

Mon

oclo

nal A

ntib

odie

s (M

abs)

Note that though clonal, Monoclonal Antibodies are (or at least used to be not)

produced by processes that havenothing to do with Gene Cloning

Pha

ge T

ypin

g

Bio

chem

ical

Tes

ts (

1/2)

Biochemical Tests (2/2)

Typ

e S

trai

n• In classifying an organism, it is helpful to

have some standard to compare it to

• Such standards for a given strain are termed type strains

• Often the type strain is the first example of a species or strain

• Type strains are kept preserved by the American Type Culture Collection (ATCC)

• (yes, I know, this is an exact quote from the lecture notes—what can I say, it was late….)

Ber

gey’

s M

anua

l

Direct Sequencing of Environmental Samples

DNA

DNA

DNA

DNA

DNA

DNA DNA

PCR

DNA DNADNA

DNA

DNA

1. Collect water orsoil sample containingbacteria and archaea.

2. Lyse cells andpurify DNA.

3. Use universal primersto amplify rRNA genesin sample by PCR.

4. Purify rRNA genes on a gel, Then separate them by inserting individual rDNAs into plasmids.

5. Insert plasmids into E.coli cells. The cells grow rapidly to produce millions of copies of each rDNA.

6. Purify rDNAfrom plasmids.

…AGGCTTACCGTAAC...

7. Sequence rDNA;compare to knownsequences.

Learning the Binomials

1. Look for Default Categories: Facultatively Anaerobic Gram-Negative Rods (Escherichia, Shigella, Salmonella, Klebsiella, Enterobacter, Serratia, Proteus, & Yersinia = Family Enterobacteriaceae; plus Gardnerella, Haemophilus, Pasteurella, & Vibrio)

2. Learn the categories: Spirochetes; Aerobic Motile helical Gram-Negative; Gram-Negative Aerobic Rods and Cocci; Anaerobic Gram-Negative Rods; Rickettias; Chlamydiaes; Mycoplasmas; Gram-Positive Cocci; Endospore-Forming Rods and Coccis, etc.

3. Start by distinguishing by Gram-staining characteristics, shape, and oxygen requirements

4. Associate disease with genera5. Associate genera with specific epithets6. Learn only that stuff not in brackets7. Don’t forget to use proper binomial-nomenclature conventions8. Start learning binomials for the previous midterm!

Chl

amyd

ia L

ife C

ycle

s

Link to Next Presentation

Ack

now

ledg

emen

ts

http://www.ulm.edu/~sdavis/b122/lectures/14.ppthttp://www.sw.vccs.edu/msht2/biology/Lecture%20in%20Power%20Point/Chapter%2022.ppthttp://www.botany.hawaii.edu/BOT201/Algae/BOT201%20CYANOPHYTE%20LECTURE%20DELIVERED.PPThttp://zadaw.com/presentations/arboviruses.pptwww.bishops.ntc.nf.ca/wells/redas/webpages/ powerpoint/Kingdoms_Taxonomy.ppthttp://www.colby.edu/biology/BI163/Bacteriappt/bacteriaarchaea.ppthttp://www.laredo.edu/science/rviswanath/BIOL1410/Chapter12.ppthttp://microbiology.okstate.edu/courses/micro2124/babus/Chapter10_files/Chapter10.ppt

DN

A H

ybrid

izat

ion

Group A Streptococcus Direct TestNucleic acid hybridization tests are based on the ability of complementary nucleic acid strands to specifically align and associate to form stable double-stranded complexes (7). The GEN-PROBE DNA Probe assay uses a single-stranded DNA probe with a chemiluminescent label which is complementary to the ribosomal RNA of the target organism. After the ribosomal RNA is released, the labeled DNA probe combines with the target organism’s ribosomal RNA to form a stable DNA:RNA hybrid. The Selection Reagent differentiates non-hybridized from hybridized probe. The labeled DNA:RNA hybrids are measured in a GEN-PROBE luminometer (LEADER® I, 50, 400, 450, or 450i). A positive result is a luminometer reading greater than or equal to the cut-off. A value below this cut-off is a negative result. http://www.gen-probe.com/PI/3890 PI.pdf

Imm

unol

ogic

al T

ests • Slide agglutination antiserum and

slides (will be doing in lab)

• Enzyme-Linked Immunosorbent Assay (ELISA) antibodies and microplates (will discuss in detail last lecture)

• Western Blotting: antigens are separated by a gel-electrophoresis and detected using radioactive or fluoresence tagged antibodies

• Along with ELISA serves as basis for assay for HIV seroconversion

Group A Streptococcus Direct TestPhadebact® Streptococcus Test is a co-agglutination test. Antibodies which are specific against streptococcal groups A, B, C and G are bound to Protein A on the surface of non-viable staphylococci. When a sample containing streptococci belonging to one of these four groups is mixed with the reagent, specific antigens on the surface of the streptococci bind to the corresponding specific antibodies. A co-agglutination lattice is formed, which is visible to the naked eye. Test time: 15 min or less. http://www.boule.se/diagnostics/10558312.htm