IV- NHU CẦU VỀ YẾU TỐ TĂNG TRƯỞNG NHU CẦU VỀ YẾU TỐ TĂNG TRƯỞNG
5. Sự thu nạp sắt (Capture de fer ) )
Hầu như mọi vi sinh vật đều cần có Fe cho hệ thống cytochrome và Hầu như mọi vi sinh vật đều cần có Fe cho hệ thống cytochrome và nhiều enzyme của nó. Việc thu nhập Fe không dễ dàng vì Fe3+ và dẫn nhiều enzyme của nó. Việc thu nhập Fe không dễ dàng vì Fe3+ và dẫn
xuất của nó khó hòa tan, nên Fe tự do thường không có sẵn cho việc xuất của nó khó hòa tan, nên Fe tự do thường không có sẵn cho việc
chuyên chở. Nhiều vi khuẩn và nấm khắc phục khó khăn này bằng cách chuyên chở. Nhiều vi khuẩn và nấm khắc phục khó khăn này bằng cách
tiết ra các phân tử vận chuyển Fe –Sidérophore.
tiết ra các phân tử vận chuyển Fe –Sidérophore.
Sidérophore Sidérophore là những chất có trọng lượng phân tử thấp, nó tập là những chất có trọng lượng phân tử thấp, nó tập hợp sidérophore (transporteur de fer
hợp sidérophore (transporteur de fer)-)-tiếng Hy Lạp có nghĩa là chất vận tiếng Hy Lạp có nghĩa là chất vận chuyển những ion Fe lại và cung cấp chúng cho tế bào.
chuyển những ion Fe lại và cung cấp chúng cho tế bào.
FerrichromeFerrichrome là một hydroxamate được sản xuất bởi một số loài là một hydroxamate được sản xuất bởi một số loài nấm; Entérobatine là một phénolates-catécholates do E. coli tiết ra. Ba nấm; Entérobatine là một phénolates-catécholates do E. coli tiết ra. Ba
nhóm của Sidérophore tương tác với những orbitale của Fe để tạo thành nhóm của Sidérophore tương tác với những orbitale của Fe để tạo thành
phức hợp octatrique có 6 nối.
phức hợp octatrique có 6 nối.
Khi có một ít Fe trong môi trường nuôi cấy, thì VSV sẽ tiết ra Khi có một ít Fe trong môi trường nuôi cấy, thì VSV sẽ tiết ra Sidérophore. Khi phức hợp Sidérophore
Sidérophore. Khi phức hợp Sidérophore--Fe đến màng tế bào, thì nó sẽ liên Fe đến màng tế bào, thì nó sẽ liên kết với protéine tiếp nhận của các sidérophore này. Kế đó Fe sẽ được
kết với protéine tiếp nhận của các sidérophore này. Kế đó Fe sẽ được phóng thích để đi trực tiếp vào trong tế bào hoặc cả phức hợp trên sẽ đi phóng thích để đi trực tiếp vào trong tế bào hoặc cả phức hợp trên sẽ đi
vào trong tế bào. Ở E. coli, chất tiếp nhận khu trú trên màng ngoài của vỏ vào trong tế bào. Ở E. coli, chất tiếp nhận khu trú trên màng ngoài của vỏ
EMB(Eosin Methylene Blue) MacConkey
Kligler Iron Agar (KIA)
Motility Indole Ornithine (MIO) Medium
Glucose Fermentation Broth
Pair of Tubes
Pair of Tubes 1st1st 2nd2nd 3rd3rd
Aerobic deamination of Aerobic deamination of
amino acids – not amino acids – not
relevant to relevant to
glucose catabolism glucose catabolism (This occurs in upper (This occurs in upper part of open tube.) part of open tube.)
++
(Blue alkaline reaction (Blue alkaline reaction
seen; not over- seen; not over- neutralized by any acid neutralized by any acid
production.) production.)
++
(Alkaline reaction (Alkaline reaction overneutralized by overneutralized by
acid.) acid.)
++
(Alkaline reaction (Alkaline reaction overneutralized by overneutralized by
acid.) acid.)
Respiration
Respiration of glucose of glucose (Acid seen in upper (Acid seen in upper part of open tube.)
part of open tube.) –– ++
This cannot This cannot
be be discerned discerned due to the due to the
high high amount of amount of
acid acid produced produced
from from fermentati fermentati
on.on.
Fermentation
Fermentation of glucos of glucos ee
(Acid diffuses
(Acid diffuses –– –– ++
Note the examples shown below. For each pair of tubes, the tube on Note the examples shown below. For each pair of tubes, the tube on the right was overlayed with mineral oil after inoculation.
the right was overlayed with mineral oil after inoculation.
First pair of tubes:First pair of tubes: Tubes were inoculated with a Tubes were inoculated with a strict aerobe which strict aerobe which neither respires nor ferments glucose
neither respires nor ferments glucose – therefore – therefore no acidic reactionno acidic reaction. . The blue alkaline reaction shows up where there is growth at the top of The blue alkaline reaction shows up where there is growth at the top of the "aerobic" tube. This is the
the "aerobic" tube. This is the negative reactionnegative reaction..
Second pair of tubes:Second pair of tubes: Tubes were inoculated with a Tubes were inoculated with a strict aerobe strict aerobe which respires but does not ferment glucose
which respires but does not ferment glucose. The small amount of . The small amount of acid associated with respiration shows up where there is growth at the acid associated with respiration shows up where there is growth at the top of the "aerobic" tube
top of the "aerobic" tube. This is the . This is the "O" reaction"O" reaction, typical for most , typical for most species of
species of PseudomonasPseudomonas. (The alkaline reaction from amino acid . (The alkaline reaction from amino acid deamination is overneutralized.)
deamination is overneutralized.)
Third pair of tubes:Third pair of tubes: Tubes were inoculated with a Tubes were inoculated with a facultative anaerobe facultative anaerobe – i.e., one which can respire (with O2) and ferment.
– i.e., one which can respire (with O2) and ferment. Acid from the Acid from the
fermentation of glucose diffuses throughout both the "aerobic" and fermentation of glucose diffuses throughout both the "aerobic" and
"anaerobic" tubes
"anaerobic" tubes. This is the . This is the "F" reaction"F" reaction, typical of the enterics. (The , typical of the enterics. (The alkaline reaction from amino acid deamination is overneutralized. Also, alkaline reaction from amino acid deamination is overneutralized. Also,
Glucose O/F Medium Glucose O/F Medium
Thioglycollate Medium
Corresponding tube no. above
Corresponding tube no. above 11 22 33 44
Oxygen relationship designation Oxygen relationship designation
STRICT STRICT
(OBLIGA (OBLIGA
TE)TE) AEROBE AEROBE
FACULTATIVE FACULTATIVE ANAEROBE
ANAEROBE AEROTOLERANTAEROTOLERANT ANAEROBE ANAEROBE
STRICT STRICT (OBL(OBL IGATIGAT E)E) ANAANA EROERO BEBE Aerobic respiration*
Aerobic respiration* ++ ++ –– ––
Fermentation*
Fermentation* –– ++ ++ ++
Ability to grow aerobically Ability to grow aerobically
(oxygen tolerance)
(oxygen tolerance) ++ ++ ++ ––
Ability to grow anaerobically
Ability to grow anaerobically –– ++ ++ ++
Catalase reaction
Catalase reaction ++ ++ –– ––
Reaction in
Reaction in Glucose O/F MediumGlucose O/F Medium
(for those able to grow well in medium)
(for those able to grow well in medium) O or –O or – FF
Response to sodium azide in a Response to sodium azide in a
growth medium
growth medium SENSITIVESENSITIVE SENSITIVE (underSENSITIVE (under aerobic aerobic conditions)
conditions) RESISTANTRESISTANT RESISTANRESISTAN TT
In Tube #1, we have a medium containing peptone and agar plus other nutrients a "typical In Tube #1, we have a medium containing peptone and agar plus other nutrients a "typical Bacteriology 102 organism" (i.e., a commonly-found, easy-to-grow chemo- or photoheterotroph) Bacteriology 102 organism" (i.e., a commonly-found, easy-to-grow chemo- or photoheterotroph) might require for metabolism and replication –
might require for metabolism and replication – except that nothing is included which would except that nothing is included which would support anaerobic growth such as glucose (or something else that could be fermented) or nitrate support anaerobic growth such as glucose (or something else that could be fermented) or nitrate (or some other electron acceptor/"oxygen substitute" that could be used in anaerobic respiration).
(or some other electron acceptor/"oxygen substitute" that could be used in anaerobic respiration).
After inoculation of this medium and incubation in the dark, any
After inoculation of this medium and incubation in the dark, any growth would be due to aerobic growth would be due to aerobic respiration
respiration with the growth with the growth only at the top of the mediumonly at the top of the medium. There would be . There would be no anaerobic no anaerobic growth
growth except for some rare, exceptional organisms which can ferment amino acids; these we do except for some rare, exceptional organisms which can ferment amino acids; these we do not consider in Bact. 102.
not consider in Bact. 102.
Tube #2 is the same medium as in #1, but glucose has been added.Tube #2 is the same medium as in #1, but glucose has been added. After incubation (in the After incubation (in the dark), any
dark), any anaerobic growth would be due to fermentation of the glucose. Thus the medium anaerobic growth would be due to fermentation of the glucose. Thus the medium can be used to detect whether or not an organism can respire (aerobically) or ferment. An example can be used to detect whether or not an organism can respire (aerobically) or ferment. An example of such a medium is the Thioglycollate Medium we use to test common chemoheterotrophs for of such a medium is the Thioglycollate Medium we use to test common chemoheterotrophs for
"oxygen relationships" (discussed
"oxygen relationships" (discussed herehere and more fully and more fully herehere).).
Tube #3 is the same medium as in #1, but potassium nitrate has been added.Tube #3 is the same medium as in #1, but potassium nitrate has been added. After After incubation (in the dark), any
incubation (in the dark), any anaerobic growth would be due to anaerobic respiration where anaerobic growth would be due to anaerobic respiration where the organism is using nitrate as the electron acceptor. In Bact. 102 (Exp. 7), we do a test in a the organism is using nitrate as the electron acceptor. In Bact. 102 (Exp. 7), we do a test in a broth medium for nitrate reduction; with reagents we can detect nitrite formation, and with the broth medium for nitrate reduction; with reagents we can detect nitrite formation, and with the Durham tube we can detect N2 gas formation. One can probably see why we would not want to Durham tube we can detect N2 gas formation. One can probably see why we would not want to include nitrate in the Thioglycollate Medium above.
include nitrate in the Thioglycollate Medium above.
Tube #4 is the same medium as in #1, but we have incubated the tube in the presence of Tube #4 is the same medium as in #1, but we have incubated the tube in the presence of light.
light. With light as the ultimate energy source, With light as the ultimate energy source, anaerobic growth would be due to anoxygenic anaerobic growth would be due to anoxygenic phototrophy
phototrophy. This is the basis for the test we do in Bact. 102 (Exp. 11.1) to see if our isolates of . This is the basis for the test we do in Bact. 102 (Exp. 11.1) to see if our isolates of purple non-sulfur bacteria are either "strict phototrophs" (just anaerobic growth in the light) or purple non-sulfur bacteria are either "strict phototrophs" (just anaerobic growth in the light) or
LEFT
LEFT Plate inoculated with 0.5 ml of sewage (which had been passed Plate inoculated with 0.5 ml of sewage (which had been passed through a filter to trap bacteria and larger particles) and
through a filter to trap bacteria and larger particles) and E. coliE. coli
strain B. Note the bacteriophage plaques appearing on the bacterial strain B. Note the bacteriophage plaques appearing on the bacterial lawn. Different phages can produce different-appearing plaques lawn. Different phages can produce different-appearing plaques (large vs. small, cloudy vs. clear, etc.). Individual plaques can be (large vs. small, cloudy vs. clear, etc.). Individual plaques can be inoculated into growing broth cultures of
inoculated into growing broth cultures of E. coliE. coli B (as one would B (as one would inoculate bacterial colonies into sterile media); thus one can
inoculate bacterial colonies into sterile media); thus one can
propagate "cultures" of bacteriophage isolates which will lyse the propagate "cultures" of bacteriophage isolates which will lyse the bacterial cells as they replicate. The resulting suspensions
bacterial cells as they replicate. The resulting suspensions ("lysates") can be filtered or chloroformed to remove any ("lysates") can be filtered or chloroformed to remove any remaining viable bacterial cells.
remaining viable bacterial cells.
RIGHRIGH Three bacteriophage isolates (I, II and III) and their activity or Three bacteriophage isolates (I, II and III) and their activity or
LEFT
LEFT Typical dry, circular, opaque, convex colonies of Typical dry, circular, opaque, convex colonies of Streptomyces Streptomyces predominate on this plate of Actinomycete Isolation Agar which predominate on this plate of Actinomycete Isolation Agar which had been inoculated with a dilution of soil. Such colonies are had been inoculated with a dilution of soil. Such colonies are
picked for future study. Shiny or filamentous colonies (some seen picked for future study. Shiny or filamentous colonies (some seen here) are not
here) are not StreptomycesStreptomyces and are therefore avoided. and are therefore avoided.
RIGHRIGH
T T The test to determine antibiotic production. After the The test to determine antibiotic production. After the Streptomyces
Streptomyces isolate (vertical streak) has grown for several days, isolate (vertical streak) has grown for several days,
Blood agar
Blood Agar: The CAMP Test Blood Agar: The CAMP Test
Streptococcus agalactiae, a member of the Streptococcus agalactiae, a member of the Lancefield Group BLancefield Group B streptococci, is one of the causative agents of
streptococci, is one of the causative agents of mastitismastitis in cows. in cows.
Identifying this organism can be difficult, and the
Identifying this organism can be difficult, and the CAMP TestCAMP Test was was
designed to aid in the identification of this organism. This test relies on designed to aid in the identification of this organism. This test relies on the fact that most
the fact that most S. agalactiaeS. agalactiae strains produce a diffusible, extracellular strains produce a diffusible, extracellular compound that will, in conjunction with a specific beta-hemolysin of
compound that will, in conjunction with a specific beta-hemolysin of Staphylococcus aureus
Staphylococcus aureus, cause complete lysis of sheep red blood cells in , cause complete lysis of sheep red blood cells in an agar medium. This test was named after the authors of the original an agar medium. This test was named after the authors of the original paper.
paper.
Remember not to confuse the terms Remember not to confuse the terms hemolysin (that which causes hemolysin (that which causes hemolysis) and
hemolysis) and hemolysishemolysis (also known as (also known as hemolytic reaction – i.e., hemolytic reaction – i.e., the reaction seen in the blood). Different Greek-letter systems apply to the reaction seen in the blood). Different Greek-letter systems apply to various hemolysins produced by
various hemolysins produced by S. aureus and hemolytic reactions.S. aureus and hemolytic reactions.
In this photo, a Blood Agar plate is shown after 24 hours of incubation at In this photo, a Blood Agar plate is shown after 24 hours of incubation at 37°C. The vertical streak is a beta-hemolysin-producing strain of
37°C. The vertical streak is a beta-hemolysin-producing strain of Staphylococcus aureus
Staphylococcus aureus, and at right angles to it are streaks of , and at right angles to it are streaks of
(1)Enterococcus faecalis(1)Enterococcus faecalis, (2), (2)Streptococcus salivariusStreptococcus salivarius, (3), (3)S. agalactiaeS. agalactiae, , and (4)
and (4)E. duransE. durans. Note the large area of complete lysis where the . Note the large area of complete lysis where the extracellular compound of
extracellular compound of S. agalactiaeS. agalactiae encounters the beta-lysin of S. encounters the beta-lysin of S.
aureus aureus..
Reference: Christie, R., N. E. Atkins, and E. Munch-Peterson. 1944. A Reference: Christie, R., N. E. Atkins, and E. Munch-Peterson. 1944. A note on a lytic phenomenon shown by group B streptococci.
note on a lytic phenomenon shown by group B streptococci. Aust. J. Exp. Aust. J. Exp.
Biol. Med. S Biol. Med. S
Beta hemolysis:Beta hemolysis: The colony is surrounded by a white or clear zone in The colony is surrounded by a white or clear zone in which few or no intact erythrocytes are found. This reaction is best seen which few or no intact erythrocytes are found. This reaction is best seen when the organism is growing under anaerobic conditions. Beta hemolysis when the organism is growing under anaerobic conditions. Beta hemolysis is caused by one or more erythrocyte-lysing enzymes called
is caused by one or more erythrocyte-lysing enzymes called hemolysins. hemolysins. In the top photo at right which shows
In the top photo at right which shows Enterococcus duransEnterococcus durans growing on growing on Blood Agar, the light to clear zones can be seen around the colonies.
Blood Agar, the light to clear zones can be seen around the colonies.
Alpha hemolysis: The colony is surrounded by a zone of intact but discolored erythrocytes that have a greenish color. This appearance is generally due to the
action of peroxide produced by the bacteria. The bottom photo shows Streptococcus mitis growing on Blood Agar; some greenish zones of alpha hemolysis are visible.
The absence of growth around an optochin disc, as seen here, helps to distinguish
Gamma hemolysis is simply a synonym for negative hemolysis in which
E. coli- lên men lactose
The modified MacConkey Agar
example
example coliformcoliform ShigellaShigella SalmonellaSalmonella Citrobacter
Citrobacter(typical)(typical) amino acids
amino acids deaminated deaminated (alkaline rx.) (alkaline rx.)
++ ++ ++
lactose lactose fermented fermented (strong acidic (strong acidic
rx.)rx.)
+ + –– ––
thiosulfate thiosulfate reduced to reduced to
HH22S (black color)S (black color) –– –– + +
net pH reaction
net pH reaction acidic (red acidic (red colony)
colony) alkaline (white alkaline (white colony)
colony)
alkaline (white alkaline (white
colony colony
plus black center plus black center
due to due to
H S)
EMB agar
Pseudomonas
Hektoen Enteric Agar
Example
Example ColiformColiform ShigellaShigella SalmonellaSalmonella Citrobacter
Citrobacter(ty(typicpic al)al)
amino acids amino acids deaminated deaminated (alkaline rx.) (alkaline rx.)
++ ++ ++
salicin salicin fermented fermented (weak acidic (weak acidic
rx.)rx.)
++ or or –– –– ––
lactose and/or lactose and/or
sucrose sucrose fermented fermented (strong acidic (strong acidic
rx.)rx.)
+ + –– ––
thiosulfate thiosulfate reduced to reduced to HH22S (black S (black
color) color)
–– –– + +
net pH reaction
net pH reaction acidic (yellow-acidic (yellow- orange)
orange) alkaline (blue-alkaline (blue- green)
green) alkaline (blue-alkaline (blue- green)
green) Click on image
Click on image
Hektoen Enteric Agar
Brilliant Green Agar Brilliant Green Agar
Example
Example ColiformColiform
Salmonella Salmonella Shigella Shigella Citrobacter
Citrobacter(typica(typica ll))
amino acids amino acids deaminated deaminated (alkaline rx.) (alkaline rx.)
++ ++
lactose and/or lactose and/or sucrose fermented sucrose fermented (strong acidic rx.)
(strong acidic rx.) + + ––
net pH reaction
net pH reaction acidic (yellow)acidic (yellow) alkaline (red)alkaline (red) Click on image
Click on image
Brilliant Green Agar
Salmonella
XLD Agar
This medium includes a relatively large amount of lysine which can be decarboxylated (an anaerobic
process), producing an alkaline product. Thus, Salmonella (lysine decarboxylation-positive) can be
differentiated from Citrobacter (lysine decarboxylation